Article

Location of the Mesopontine Neurons Responsible for Maintenance of Anesthetic Loss of Consciousness

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Abstract

The transition from wakefulness to general anesthesia is widely attributed to suppressive actions of anesthetic molecules distributed by the systemic circulation to the cerebral cortex (for amnesia and loss of consciousness) and to the spinal cord (for atonia and antinocice-ption). An alternative hypothesis proposes that anesthetics act on one or more brainstem or diencephalic nuclei, with suppression of cortex and spinal cord mediated by dedicated axonal pathways. Previously, we documented induction of an anesthesia-like state in rats by microinjection of small amounts of GABAA-receptor agonists into an upper brainstem region named the mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesioning this area rendered animals resistant to systemically delivered anesthetics. Here, using rats of both sexes, we applied a modified microinjection method that permitted localization of the anesthetic-sensitive neurons with much improved spatial resolution. Microinjected at the MPTA hotspot identified, exposure of 1900 or fewer neurons to muscimol was sufficient to sustain whole-body general anesthesia; microinjection as little as 0.5 mm off-target did not. The GABAergic anesthetics pentobarbital and propofol were also effective. The GABA-sensitive cell cluster is centered on a tegmental (reticular) field traversed by fibers of the superior cerebellar peduncle. It has no specific nuclear designation and has not previously been implicated in brain-state transitions.

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... Group data for the open field test ( Figure, panel B) show that in control mice, the total distance traveled was similar after saline (12.7 ± 4.1 m) versus CNO (11.0 ± 4.5 m). However, in hM3Dq mice, the total distance was 18.1 ± 7.1 m after saline and 3.7 ± 4.5 m after CNO (group × treatment interaction, F [1,16] = 25.0, P = .0001). ...
... In control mice, no practical difference was observed after saline (1.65% ± 0.21%) versus CNO (1.54% ± 0.17%). In hM3Dq mice, however, the inhaled sevoflurane concentration required to induce LORR was 1.65% ± 0.25% after saline and 1.00% ± 0.27% after CNO (group × treatment interaction, F [1,16] = 18.4; P = .0006). ...
... It has been reported that microinjections of GABAergic anesthetics such as pentobarbital and propofol in the mesopontine tegmental anesthesia area (MPTA) produces a state of unconsciousness akin to general anesthesia in rodents, 16 and lesioning MPTA neurons produces resistance to these anesthetics. 17 It is possible that the sedation and increased sensitivity to sevoflurane observed in our study was mediated, at least in part, by increased levels of GABA in the MPTA. ...
Article
Many general anesthetics potentiate gamma-aminobutyric acid (GABA) A receptors but their neuroanatomic sites of action are less clear. GABAergic neurons in the rostromedial tegmental nucleus (RMTg) send inhibitory projections to multiple arousal-promoting nuclei, but the role of these neurons in modulating consciousness is unknown. In this study, designer receptors exclusively activated by designer drugs (DREADDs) were targeted to RMTg GABAergic neurons of Vgat-ires-Cre mice. DREADDs expression was found in the RMTg and other brainstem regions. Activation of these neurons decreased movement and exploratory behavior, impaired motor coordination, induced electroencephalogram (EEG) oscillations resembling nonrapid eye movement (NREM) sleep without loss of righting and reduced the dose requirement for sevoflurane-induced unconsciousness. These results suggest that GABAergic neurons in the RMTg and other brainstem regions promote sedation and facilitate sevoflurane-induced unconsciousness.
... A significant step forward was the discovery in rats that selective delivery of minute quantities of GABA A -receptor (GABA A -R) agonists into a small brainstem nucleus, the mesopontine tegmental anesthesia area (MPTA), rapidly and reversibly induces a state of general anesthesia. Conversely, lesioning this area does not cause coma as might be expected ), but rather renders rats relatively insensitive to anesthetic induction by GABAergic agents delivered systemically (Devor and Zalkind 2001;Voss et al. 2005;Namjoshi et al. 2009;Minert and Devor 2016;Sukhotinsky et al. 2016;Minert et al. 2017Minert et al. , 2020. Analysis of the mechanisms whereby activating GABA A -Rs in the MPTA induces anesthesia in rats has proceeded apace. ...
... Our initial approach to delivering very small volumes of drugs to the brainstem in rats involved use a permanently implanted cannula (Devor and Zalkind 2001). As this method proved impractical in mice due to their small size we instead adopted the alternative "bonus-time" method (Minert et al. 2017). In this method, the animal is initially anesthetized with a bolus dose of systemic propofol and subsequently, the GABA A -R agonist muscimol is microinjected into the putative MPTA using a stereotactic device. ...
... In rats, 10 mg/kg iv propofol induced highly stable baseline anesthesia times lasting 11 ± 2 min (± the standard deviation (SD). "On-target" microinjections of muscimol reliably prolonged this time, often by 10s of minutes (Minert et al. 2017). We began experiments in mice using the same propofol dose delivered via a pediatric catheter inserted into a tail vein under brief isoflurane sedation (Neoflon 26 g, Becton-Dickinson, Helsingborg, Sweden). ...
Article
Full-text available
The mesopontine tegmental anesthesia area (MPTA) was identified in rats as a singular brainstem locus at which microinjection of minute quantities of GABAergic agents rapidly and reversibly induces loss-of-consciousness and a state of general anesthesia, while lesioning renders animals insensitive to anesthetics at normal systemic doses. Obtaining similar results in mice has been challenging, however, slowing research progress on how anesthetics trigger brain-state transitions. We have identified roadblocks that impeded translation from rat to mouse and tentatively located the MPTA equivalent in this second species. We describe here a series of modifications to the rat protocol that allowed us to document pro-anesthetic changes in mice following localized stereotactic delivery of minute quantities (20 nL) of the GABAA-receptor agonist muscimol into the brainstem mesopontine tegmentum. The optimal locus identified proved to be homologous to the MPTA in rats, and local neuronal populations in rats and mice were similar in size and shape. This outcome should facilitate application of the many innovative gene-based methodologies available primarily in mice to the study of how activity in brainstem MPTA neurons brings about anesthetic loss-of-consciousness and permits pain-free surgery.
... One study by Minert et al. 22 examined the role of an upper brainstem region, the mesopontine tegmental area, in anesthetic-induced unconsciousness. They reported that microinjection of the GABA A -potentiating anesthetics propofol and pentobarbital, as well as the GABA A agonist muscimol, into the mesopontine tegmental area resulted in sustained unconsciousness. ...
... Therefore, resulting changes in physiology or behavior may operate through different mechanisms than systemic doses. For example, both Gamou et al. 24 and Minert et al. 22 are examples of studies that used supratherapeutic doses in their microinjections; an awareness of this context is necessary for accurate interpretation of these studies. (In contrast, because of the constant perfusion of fluid with microdialysis, it is not necessary to use drug concentrations higher than the target concentration.) ...
... Anesthesiology 2020; 133:[19][20][21][22][23][24][25][26][27][28][29][30] Melonakos et al. ...
Article
The neural circuits underlying the distinct endpoints that define general anesthesia remain incompletely understood. It is becoming increasingly evident, however, that distinct pathways in the brain that mediate arousal and pain are involved in various endpoints of general anesthesia. To critically evaluate this growing body of literature, familiarity with modern tools and techniques used to study neural circuits is essential. This Readers' Toolbox article describes four such techniques: (1) electrical stimulation, (2) local pharmacology, (3) optogenetics, and (4) chemogenetics. Each technique is explained, including the advantages, disadvantages, and other issues that must be considered when interpreting experimental results. Examples are provided of studies that probe mechanisms of anesthesia using each technique. This information will aid researchers and clinicians alike in interpreting the literature and in evaluating the utility of these techniques in their own research programs.
... 6,7,[11][12][13][14][15] Using a systematic microinjection approach, we advanced the dedicated pathways idea with the discovery of a small nucleus, the mesopontine tegmental anesthesia area, which appears to be a key node in circuitry that enables anesthetic induction. [16][17][18] Briefly, we showed the following: (1) Delivering the γ-aminobutyric acid-mediated (GABAergic) anesthetics pentobarbital and propofol into the mesopontine tegmental anesthesia area, or the specific GABA A -receptor agonist muscimol, quickly and reversibly induces an anesthesia-like state closely resembling systemically induced general anesthesia (http://links. lww.com/AA/B466). ...
... The minute doses used and the rapid onset time preclude the possibility of agent redistribution to the spinal cord and cortex. 18 (2) Lesions of this area reduce the sensitivity of rats to systemic pentobarbital such that anesthesia can no longer be induced at clinically relevant doses. 19 Because the rest of the CNS continues to be exposed to normal anesthetic concentrations of the pentobarbital, but anesthesia is not induced, this observation challenges the hypothesis of global or patch-wise suppression. ...
... The second was the bonus-time method. 18 Here, animals were transiently anesthetized with an iv bolus dose of 1% propofol. Then just before the anticipated time of emergence, 50 to 200 nl of alfaxalone (10 mg/ml) were microinjected into the mesopontine tegmental anesthesia area through a fine glass pipette. ...
Article
What we already know about this topic: Lesions of the mesopontine tegmental anesthesia area in the brainstem render rats strongly insensitive to pentobarbitalThe effects of mesopontine tegmental anesthesia area lesions on responses to other anesthetics have not been previously reported WHAT THIS ARTICLE TELLS US THAT IS NEW: Targeted microinjection of ibotenic acid into the mesopontine tegmental anesthesia area in adult rats led to an up to twofold loss in anesthetic potency of etomidate and propofolIn contrast, the potency of ketamine, medetomidine, and alfaxolone/alfadolone was unaffectedThese observations suggest that the mesopontine tegmental anesthesia area of the brainstem may serve as a key structure to selectively mediate transition from wakefulness into an anesthetic state in response to γ-aminobutyric acid-mediated anesthetics BACKGROUND:: The brainstem mesopontine tegmental anesthesia area is a key node in circuitry responsible for anesthetic induction and maintenance. Microinjecting the γ-aminobutyric acid-mediated (GABAergic) anesthetic pentobarbital in this nucleus rapidly and reversibly induces general anesthesia, whereas lesioning it renders the animal relatively insensitive to pentobarbital administered systemically. This study investigated whether effects of lesioning the mesopontine tegmental anesthesia area generalize to other anesthetic agents. Methods: Cell-selective lesions were made using ibotenic acid, and rats were later tested for changes in the dose-response relation to etomidate, propofol, alfaxalone/alfadolone, ketamine, and medetomidine delivered intravenously using a programmable infusion pump. Anesthetic induction for each agent was tracked using five behavioral endpoints: loss of righting reflex, criterion for anesthesia (score of 11 or higher), criterion for surgical anesthesia (score of 14 or higher), antinociception (loss of pinch response), and deep surgical anesthesia (score of 16). Results: As reported previously for pentobarbital, on-target mesopontine tegmental anesthesia area lesions reduced sensitivity to the GABAergic anesthetics etomidate and propofol. The dose to achieve a score of 16 increased to 147 ± 50% of baseline in control animals ± SD (P = 0.0007; 7 lesioned rats and 18 controls) and 136 ± 58% of baseline (P = 0.010; 6 lesioned rats and 21 controls), respectively. In contrast, responsiveness to the neurosteroids alfaxalone and alfadolone remained unchanged compared with baseline (94 ± 24%; P = 0.519; 6 lesioned rats and 18 controls) and with ketamine increased slightly (90 ± 11%; P = 0.039; 6 lesioned rats and 19 controls). The non-GABAergic anesthetic medetomidine did not induce criterion anesthesia even at the maximal dose tested. The dose to reach the maximal anesthesia score actually obtained was unaffected by the lesion (112 ± 8%; P = 0.063; 5 lesioned rats and 18 controls). Conclusions: Inability to induce anesthesia in lesioned animals using normally effective doses of etomidate, propofol, and pentobarbital suggests that the mesopontine tegmental anesthesia area is the effective target of these, but not necessarily all, GABAergic anesthetics upon systemic administration. Cortical and spinal functions are likely suppressed by recruitment of dedicated ascending and descending pathways rather than by direct, distributed drug action.
... The MPTA is flanked by the ventrolateral periaqueductal gray (PAG), reticular tegmental nucleus of the pons, cholinergic pedunculopontine tegmental nucleus (PPN), and median and paramedian pontine raphe [82]. MPTA is sensitive to GABAergic anesthetics, including pentobarbital, propofol, and etomidate [81,83,84]. Systemic administration of the classical GABAergic anesthetic pentobarbital (50 mg/kg) can suppress c-fos expression in the MPTA, indicating that neuronal activity of MPTA may be inhibited during GABAergic agent-induced anesthesia [19]. ...
... These inhibitory effects of classical GABAergic anesthetics may be due to the direct activation of postsynaptic GABA A receptors. Indeed, localized injections of pentobarbital into the MPTA can induce a complete state of anesthesia, which appears to be mediated by a circuit of dedicated axonal projections to the nearby arousal nuclei of the brainstem and distant targets in the forebrain and spinal cord [81,84,85]; meanwhile, lesions in this region lead to insomnia [86]. These results provide direct evidence for the role of subcortical regions in mediating GABAergic anesthetic-induced unconsciousness, and MPTA may serve as the on-off switch of GABAergic anesthetic-induced anesthesia. ...
Article
Although general anesthetics have been used in the clinic for more than 170 years, the ways in which they induce amnesia, unconsciousness, analgesia, and immobility remain elusive. Modulations of various neural nuclei and circuits are involved in the actions of general anesthetics. The expression of the immediate early gene c-fos and its nuclear product, c-fos protein can be induced by neuronal depolarization; therefore, c-fos staining is commonly used to identify the activated neurons during sleep and/or wakefulness, as well as in various physiological conditions in the central nervous system. Identifying c-fos expression is also a direct and convenient method to explore the effects of general anesthetics on the activity of neural nuclei and circuits. Using c-fos staining, general anesthetics have been found to interact with sleep- and wakefulness-promoting systems throughout the brain, which may explain their ability to induce unconsciousness and emergence from general anesthesia. This review summarizes the actions of general anesthetics on neural nuclei and circuits based on c-fos expression.
... Existing strategies to address complexities of anesthetic effects on higher-order structures such as circuits and networks are mostly comprised of local administrations by microinjection or infusions of anesthetics at specific structures in the central nervous system(CNS). [1][2][3] This strategy accounts for anesthetic effects possibly arising from combined effects on multiple neuronal types and has previously proven relatively effective. The primary disadvantages of this approach are poor control of concentrations, and diffusion of anesthetic away from the area of investigation resulting in effects from drug at areas external to the region of interest (particularly relevant if the area under investigation is near to a ventricle.) ...
Chapter
Investigation of how anesthetics produce hypnosis requires knowledge of their effects at the molecular, neuronal, circuit, and whole-brain network level. Anesthetic photolabels have long been used to explore how anesthetics bind and affect known protein targets, but they could potentially assist in investigation of anesthetic effects at higher organizational levels of the central nervous system. Here, we advocate the use and provide detailed methods for the application of anesthetic photolabels in slice electrophysiology and in intact animals as a means of investigating anesthetic effects on distinct circuits and brain centers.
... Although neither the precise neurochemical identities nor the electrophysiological properties of wake-promoting pontine reticular formation neurons are known, the contribution of this region to sleep and wakefulness has long been recognized [121]. Localized microinjections of GABAergic anesthetic drugs into a cluster of several thousand mesopontine tegmental neurons in the pontine reticular formation induces a complete state of general anesthesia in rodents [122]. Lesioning neurons in this region also increases wakefulness in rats at the expense of NREM and REM sleep [123]. ...
Article
Full-text available
General anesthesia serves a critically important function in the clinical care of human patients. However, the anesthetized state has foundational implications for biology because anesthetic drugs are effective in organisms ranging from paramecia, to plants, to primates. Although unconsciousness is typically considered the cardinal feature of general anesthesia, this endpoint is only strictly applicable to a select subset of organisms that are susceptible to being anesthetized. We review the behavioral endpoints of general anesthetics across species and propose the isolation of an organism from its environment - both in terms of the afferent arm of sensation and the efferent arm of action - as a generalizable definition. We also consider the various targets and putative mechanisms of general anesthetics across biology and identify key substrates that are conserved, including cytoskeletal elements, ion channels, mitochondria, and functionally coupled electrical or neural activity. We conclude with a unifying framework related to network function and suggest that general anesthetics - from single cells to complex brains - create inefficiency and enhance modularity, leading to the dissociation of functions both within an organism and between the organism and its surroundings. Collectively, we demonstrate that general anesthesia is not restricted to the domain of modern medicine but has broad biological relevance with wide-ranging implications for a diverse array of species.
... Direct electrical stimulation at appropriate subcortical locations in humans and animals readily evokes pain experience and pain behavior, subcortical seizure activity can be painful, appropriate subcortical regions are activated in functional imaging and pathway lesions can relieve pain [31,56,95]. What is more, localized microinjection of minute quantities of opiates into the brainstem PAG yields selective whole-body analgesia and microinjecting GABAergic anesthetics into the brainstem MPTA (mesopontine tegmental anesthesia area) yields LOC including insensitivity to noxious stimuli [96][97][98][99]. Most telling of all, localized lesions in the dorsal mesopontine tegmentum readily induce LOC in animals and humans, and lesions limited to the MPTA render animals relatively insensitive to otherwise clinically effective doses of anaesthetics delivered systemically [90,91,[100][101][102]. ...
Article
Full-text available
Doubtless, the conscious brain integrates masses of information. But declaring that consciousness simply "emerges" when enough has accumulated, doesn't really explain how first person experience is implemented by neurons. Moreover, empirical observations challenge integrated information theory's (IIT) reliance on thalamo-cortical interactions as the information integrator. More likely, the cortex streams processed information to a still-enigmatic consciousness generator, one perhaps located in the brainstem.
... Despite its fundamental importance, the neural circuit and network mechanisms underlying LOC have remained unclear. Several brain areas have been implicated in causing the loss or recovery of consciousness, such as the hypothalamus (Herrera et al., 2016), thalamus (Castaigne et al., 1981), basal ganglia and claustrum (Crick and Koch, 2005;Mhuircheartaigh et al., 2010), or the brain stem (Minert et al., 2017;Moruzzi and Magoun, 1949;Penfield, 1954). Although thalamocortical connections have been central to research on LOC (Flores et al., 2017;Herrera et al., 2016;Penfield, 1954;Steriade, 2003), the role of the cerebral cortex itself remains controversial (Merker, 2007). ...
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Medically-induced loss of consciousness (mLOC) has been linked to a macroscale break-down of brain connectivity, yet the neural microcircuit correlates of mLOC remain unknown. We applied non-linear t-stochastic neighbor embedding (t-SNE) and Lempel-Ziv-Welch complexity analysis to two-photon calcium imaging and local field potential (LFP) measurements of cortical microcircuit activity across anesthetic depth in mice, and to micro-electrode array recordings in human subjects. We find that mLOC disrupts population activity patterns by i) a reduction of discriminable network microstates and ii) a reduction of independent neuronal ensembles. These alterations are not explained by a simple reduction of neuronal activity and reveal abnormal functional microcircuits. Thus, normal neuronal ensemble dynamics could contribute to the emergence of conscious states.
... Finding the biological basis of consciousness represents a challenging and outstanding open problem for science [4]. Different brain regions such as brain stem [5][6][7][8], basal ganglia [9,10], claustrum [11], or the hypothalamus [12]have been involved in the loss or restoration of consciousness. Although thalamocortical circuits represent critical enabling factors for consciousness [13][14][15][16][17][18], the importance of the different cortical areas in supporting consciousness is not completely elucidated [2,3,[19][20][21][22][23][24][25][26]. ...
Article
Recent studies suggest that vagus nerve stimulation (VNS) promotes cognitive and behavioral restoration after traumatic brain injuries. As vagus nerve has wide effects over the brain and visceral organs, stimulation of the sensory/visceral afferents might have a therapeutic potential to modulate the level of consciousness. One of the most important challenges in studying consciousness is objective evaluation of the consciousness level. Brain complexity that can be measured through Lempel-Ziv complexity (LZC) index was used as a novel mathematical approach for objective measurement of consciousness. The main goal of our study was to examine the effects of VNS on LZC index of consciousness. In this study, we did VNS on the anesthetized rats, and simultaneously LFPs recording was performed in two different cortical areas of primary somatosensory (S1) or visual (V1) cortex. LZC and the amplitude of slow waves were computed during different periods of VNS. We found that the LZC index during VNS period was significantly higher in both of the cortical areas of S1 and V1. Slow-wave activity decreased during VNS in S1, while there was no significant change in V1. Our findings showed that VNS can augment the consciousness level, and LZC index is a more sensitive parameter for detecting the level of consciousness.
... Subcortical action is further supported by microinjection experiments showing that highly localized drug application to some mesopontine/midbrain regions can, on their own and without direct cortical effects, cause an anesthesialike state, 18,35,36 as well as studies showing that stimulation of the dopaminergic ventral tegmental area can hasten arousal from anesthesia in rodents. 37 Minert et al. 38 have shown that anesthetic microinjection into a highly localized region of the upper brainstem called the mesopontine tegmental anesthetic area induces a reversible state of unconsciousness, along with associated anesthesia-like changes in the electroencephalogram. 35 Investigation of fos protein expression suggests that general anesthetic cortical action may be attributable to effects at subcortical neuromodulatory sites. 39 It should be noted, however, that these results have not been fully replicated elsewhere. ...
Article
General anesthetics have been used to ablate consciousness during surgery for more than 150 yr. Despite significant advances in our understanding of their molecular-level pharmacologic effects, comparatively little is known about how anesthetics alter brain dynamics to cause unconsciousness. Consequently, while anesthesia practice is now routine and safe, there are many vagaries that remain unexplained. In this paper, the authors review the evidence that cortical network activity is particularly sensitive to general anesthetics, and suggest that disruption to communication in, and/or among, cortical brain regions is a common mechanism of anesthesia that ultimately produces loss of consciousness. The authors review data from acute brain slices and organotypic cultures showing that anesthetics with differing molecular mechanisms of action share in common the ability to impair neurophysiologic communication. While many questions remain, together, ex vivo and in vivo investigations suggest that a unified understanding of both clinical anesthesia and the neural basis of consciousness is attainable.
... Direct electrical stimulation at appropriate subcortical locations in humans and animals readily evokes pain experience and pain behavior, subcortical seizure activity can be painful, appropriate subcortical regions are activated in functional imaging and pathway lesions can relieve pain [31,56,95]. What is more, localized microinjection of minute quantities of opiates into the brainstem PAG yields selective whole-body analgesia and microinjecting GABAergic anesthetics into the brainstem MPTA (mesopontine tegmental anesthesia area) yields LOC including insensitivity to noxious stimuli [96][97][98][99]. Most telling of all, localized lesions in the dorsal mesopontine tegmentum readily induce LOC in animals and humans, and lesions limited to the MPTA render animals relatively insensitive to otherwise clinically effective doses of anaesthetics delivered systemically [90,91,[100][101][102]. ...
Article
Full-text available
It is nearly axiomatic that pain, among other examples of conscious experience, is an outcome of still-uncertain forms of neural processing that occur in the cerebral cortex, and specifically within thalamo-cortical networks. This belief rests largely on the dramatic relative expansion of the cortex in the course of primate evolution, in humans in particular, and on the fact that direct activation of sensory representations in the cortex evokes a corresponding conscious percept. Here we assemble evidence, drawn from a number of sources, suggesting that pain experience is unlike the other senses and may not, in fact, be an expression of cortical processing. These include the virtual inability to evoke pain by cortical stimulation, the rarity of painful auras in epileptic patients and outcomes of cortical lesions. And yet, pain perception is clearly a function of a conscious brain. Indeed, it is perhaps the most archetypical example of conscious experience. This draws us to conclude that conscious experience, at least as realized in the pain system, is seated subcortically, perhaps even in the “primitive” brainstem. Our conjecture is that the massive expansion of the cortex over the course of evolution was not driven by the adaptive value of implementing consciousness. Rather, the cortex evolved because of the adaptive value of providing an already existing subcortical generator of consciousness with a feed of critical information that requires the computationally intensive capability of the cerebral cortex.
... Secondly, the illustrations of bilateral injection sites are shown in Fig. 5c-e for each treatment animal. The modest volume of drugs and saline (0.25 µl) used in our study were slightly less [27] or more [28] than used in other studies where the drugs diffusion a Typical examples of the EEG/EMG recordings and the corresponding hypnograms after administration of saline or bicuculline at 0.25 μg/side for 10 h (from 9:00 to 19:00). b 12 h time-course changes in wakefulness, NREM, and REM sleep in rats treated with bicuculline at 0.25 μg/side. ...
Article
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GABA, the most abundant inhibitory neurotransmitter in the brain, is closely linked with sleep and wakefulness. As the largest area input to the ventral pallidum (VP), the nucleus accumbens (NAc) has been confirmed to play a pivotal role in promoting non-rapid eye movement (NREM) sleep through inhibitory projections from NAc adenosine A2A receptor-expressing neurons to VP GABAergic neurons which mostly express GABAA receptors. Although these studies demonstrate the possible role of VP GABAergic neurons in sleep–wake regulation, whether and how its modulate sleep–wake cycle is not completely clear. In our study, pharmacological manipulations were implemented in freely moving rats and then the EEG and the EMG were recorded to monitor the sleep–wake states. We found that microinjection of muscimol, a GABAA receptor agonist, into the VP increased NREM sleep in both light and dark period. Microinjection of bicuculline, a GABAA receptor antagonist, into the VP increased wakefulness in the light period. Collectively, our data identify the important role of VP GABAA receptor-expressing neurons in NREM sleep of rats which may help improve the understanding of the pathological sleep disorders.
... Despite its fundamental importance, the neural circuit and network mechanisms underlying LOC have remained unclear. Several brain areas have been implicated in causing the loss or recovery of consciousness, such as the hypothalamus (Herrera et al., 2016), thalamus (Castaigne et al., 1981), basal ganglia and claustrum (Crick and Koch, 2005;Mhuircheartaigh et al., 2010), or the brain stem (Minert et al., 2017;Moruzzi and Magoun, 1949;Penfield and Jasper, 1954). Although thalamocortical connections have been central in LOC research (Flores et al., 2017;Herrera et al., 2016;Penfield and Jasper, 1954;Steriade, 2003), the role of the cortex itself remains debated (Merker, 2007). ...
Article
Full-text available
Medically induced loss of consciousness (mLOC)during anesthesia is associated with a macroscale breakdown of brain connectivity, yet the neural microcircuit correlates of mLOC remain unknown. To explore this, we applied different analytical approaches (t-SNE/watershed segmentation, affinity propagation clustering, PCA, and LZW complexity)to two-photon calcium imaging of neocortical and hippocampal microcircuit activity and local field potential (LFP)measurements across different anesthetic depths in mice, and to micro-electrode array recordings in human subjects. We find that in both cases, mLOC disrupts population activity patterns by generating (1)fewer discriminable network microstates and (2)fewer neuronal ensembles. Our results indicate that local neuronal ensemble dynamics could causally contribute to the emergence of conscious states.
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General anesthesia provides an invaluable experimental tool to probe the essential neural circuits that underlie consciousness. A new study reports that cholinergic stimulation of the prefrontal cortex restores wake-like behaviors in anesthetized rodents, suggesting that cholinergic inputs to the prefrontal cortex play a fundamental role in modulating consciousness.
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Cambridge Core - Psychiatry and Clinical Psychology - How Brain Arousal Mechanisms Work - by Donald Pfaff
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We all experience pain at one time or another. Pain is an essential “alarm bell” that tells us that something is wrong, and a “teacher” that reminds us not to do that same thing again. Usually, pain is felt when a stimulus, such as a pinch or an injury, causes electrical pulses to run along one of the cables of nerve fibers in our body and into the brain where they generate an unpleasant sensory and emotional experience. Sometimes pain occurs without any actual stimulus, such as when nerve fibers have been damaged. An example is the phantom pain that amputees sometimes feel in their missing limb. Certain drugs can stop pain by blocking the electrical pulses before they reach the brain. Other drugs stop pain in a different way, by preventing the brain from reading the pain message carried by the electrical pulses. This article explains what happens in the body when we are hurt, how the brain causes this to be felt as pain and how certain drugs can stop pain.
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The mesopontine tegmental anesthesia area (MPTA) is a small brainstem nucleus that, when exposed to minute quantities of GABAA receptor agonists, induces a state of general anesthesia. In addition to immobility and analgesia this state is accompanied by widespread suppression of neural activity in the cerebral cortex and high delta-band power in the electroencephalogram. Collectively, MPTA neurons are known to project to a variety of forebrain targets which are known to relay to the cortex in a highly distributed manner. Here we ask whether ascending projections of individual MPTA neurons collateralize to several of these cortical relay nuclei, or access only one. Using rats, contrasting retrograde tracers were microinjected pairwise on one side into three ascending relays: the basal forebrain, the zona incerta-lateral hypothalamus and the intralaminar thalamic nuclear group. In addition, in separate animals, each target was microinjected bilaterally. MPTA neurons were then identified as being single-or double-labeled, indicating projection to one target nucleus or collateralization to both. Results indicated that double-labeling was rare, occurring on average in only 1.3% of the neurons sampled. The overwhelming majority of individual MPTA neurons showed specific connectivity, contributing to only one of the major ascending pathways, either ipsilaterally or contralaterally, but not bilaterally. This architecture would permit particular functional aspects of anesthetic loss-of-consciousness to be driven by specific subpopulations of MPTA neurons.
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In light of the general shift from rats to mice as the leading rodent model in neuroscience research we used c-Fos expression as a tool to survey brain regions in the mouse in which neural activity differs between the states of wakefulness and pentobarbital-induced general anesthesia. The aim was to complement prior surveys carried out in rats. In addition to a broad qualitative review, 28 specific regions of interest (ROIs) were evaluated quantitatively. Nearly all ROIs in the cerebral cortex showed suppressed activity. Subcortically, however, some ROIs showed suppression, some showed little change, and some showed increased activity. The overall picture was similar to the rat. Special attention was devoted to ROIs significantly activated during anesthesia as such loci might actively drive the transition to anesthetic unconsciousness rather than responding passively to inhbitory agents distributed globally (the “wet blanket” hypothesis). Twelve such “anesthesia-on” ROIs were identified: the paraventricular hypothalamic nucleus, supraoptic nucleus, tuberomamillary nucleus, lateral habenular nucleus, dentate gyrus, nucleus raphe pallidus, central amygdaloid nucleus, perifornical lateral hypothalamus, ventro-lateral preoptic area, lateral septum, paraventricular thalamic nucleus and zona incerta. The same primary anti-FOS antibody was used in all mice, but two alternative reporter systems were employed: ABC-diaminobenzidine and the currently more popular AlexaFluor488. Fluorescence tagging revealed far fewer FOS-immunoreactive neurons, sounding an alert that the reporter system chosen can have major effects on results obtained.
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In order to understand general anaesthesia and certain seizures, a fundamental understanding of the neurobiology of unconsciousness is needed. This article explores similarities in neuronal and network changes during general anaesthesia and seizure-induced unconsciousness. Both seizures and anaesthetics cause disruption in similar anatomical structures that presumably lead to impaired consciousness. Despite differences in behaviour and mechanisms, both of these conditions are associated with disruption of the functionality of subcortical structures that mediate neuronal activity in the frontoparietal cortex. These areas are all likely to be involved in maintaining normal consciousness. An assessment of the similarities in the brain network disruptions with certain seizures and general anaesthesia might provide fresh insights into the mechanisms of the alterations of consciousness seen in these particular unconscious states, allowing for innovative therapies for seizures and the development of anaesthetic approaches targeting specific networks.
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General anesthetic agents are thought to induce loss-of-consciousness (LOC) and enable pain-free surgery by acting on the endogenous brain circuitry responsible for sleep-wake cycling. In clinical use, the entire CNS is exposed to anesthetic molecules with LOC usually attributed to synaptic suppression in the cerebral cortex and immobility and analgesia to agent action in the spinal cord and brainstem. This model of patch-wise suppression has been challenged, however, by the observation that all functional components of anesthesia can be induced by focal delivery of minute quantities of GABAergic agonists to the brainstem mesopontine tegmental anesthesia area (MPTA). We compared spectral features of the cortical electroencephalogram (EEG) in rats during systemic anesthesia and anesthesia induced by MPTA microinjection. Systemic administration of (GABAergic) pentobarbital yielded the sustained, δ-band dominant EEG signature familiar in clinical anesthesia. In contrast, anesthesia induced by MPTA microinjection (pentobarbital or muscimol) featured epochs of δ-band EEG alternating with the wake-like EEG, the pattern typical of natural non-rapid-eye-movement (NREM) and REM sleep. The rats were not sleeping, however, as they remained immobile, atonic and unresponsive to noxious pinch. Recalling the paradoxical wake-like quality the EEG during REM sleep, we refer to this state as “paradoxical anesthesia”. GABAergic anesthetics appear to co-opt both cortical and spinal components of the sleep network via dedicated axonal pathways driven by MPTA neurons. Direct drug exposure of cortical and spinal neurons is not necessary, and is probably responsible for off-target side-effects of systemic administration including monotonous δ-band EEG, hypothermia and respiratory depression. Significance statement The concept that GABAergic general anesthetic agents induce loss-of-consciousness by substituting for an endogenous neurotransmitter, thereby co-opting neural circuitry responsible for sleep-wake transitions, has gained considerable traction. However, the electroencephalographic (EEG) signatures of sleep and anesthesia differ fundamentally. We show that when the anesthetic state is generated by focal delivery of GABAergics into the mesopontine tegmental anesthesia area (MPTA) the resulting EEG repeatedly transitions between delta-wave-dominant and wake-like patterns much as in REM-NREM sleep. This suggests that systemic (clinical) anesthetic delivery, which indiscriminately floods the entire cerebrum with powerful inhibitory agents, obscures the sleep-like EEG signature associated with the less adulterated form of anesthesia obtained when the drugs are applied selectively to loci where the effective neurotransmitter substitution actually occurs.
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The brain undergoes rapid, dramatic, and reversible transitioning between states of wakefulness and unconsciousness during natural sleep and in pathological conditions such as hypoxia, hypotension, and concussion. Transitioning can also be induced pharmacologically using general anesthetic agents. The effect is selective. Mobility, sensory perception, memory formation, and awareness are lost while numerous housekeeping functions persist. How is selective transitioning accomplished? Classically a handful of brainstem and diencephalic “arousal nuclei” have been implicated in driving brain-state transitions on the grounds that their net activity systematically varies with brain state. Here we used transgenic targeted recombination in active populations mice
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We review evidence challenging the hypothesis that memories are processed or consolidated in sleep. We argue that the brain is in an unconscious state in sleep, akin to general anesthesia, and hence is incapable of meaningful cognitive processing – the sole purview of waking consciousness. At minimum, the encoding of memories in sleep would require that waking events are faithfully transferred to and reproduced in sleep. Remarkably, however, this has never been demonstrated, as waking experiences are never truly replicated in sleep but rather appear in very altered or distorted forms. General anesthetics (GAs) exert their effects through endogenous sleep‐wake control systems and accordingly GAs and sleep share several common features: sensory blockade, immobility, amnesia and lack of awareness (unconsciousness). The loss of consciousness in non‐REM (NREM) sleep or to GAs is characterized by: (1) delta oscillations throughout the cortex; (2) marked reductions in neural activity (from waking) over widespread regions of the cortex, most pronounced in frontal and parietal cortices; and (3) a significant disruption of the functional connectivity of thalamocortical and corticocortical networks, particularly those involved in “higher order” cognitive functions. Several (experimental) reports in animals and humans have shown that disrupting the activity of the cortex, particularly the orbitofrontal cortex, severely impairs higher order cognitive and executive functions. The profound and widespread deactivation of the cortex in the unconscious states of NREM sleep or GA would be expected to produce an equivalent, or undoubtedly a much greater, disruptive effect on mnemonic and cognitive functions. In conclusion, we contend that the unconscious, severely altered state of the brain in NREM sleep would negate any possibility of cognitive processing in NREM sleep. This article is protected by copyright. All rights reserved.
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Dopamine (DA) promotes wakefulness, and DA transporter inhibitors such as dextroamphetamine and methylphenidate are effective for increasing arousal and inducing reanimation, or active emergence from general anesthesia. DA neurons in the ventral tegmental area (VTA) are involved in reward processing, motivation, emotion, reinforcement, and cognition, but their role in regulating wakefulness is less clear. The current study was performed to test the hypothesis that selective optogenetic activation of VTA DA neurons is sufficient to induce arousal from an unconscious, anesthetized state. Floxed-inverse (FLEX)-Channelrhodopsin2 (ChR2) expression was targeted to VTA DA neurons in DA transporter (DAT)-cre mice (ChR2+ group; n = 6). Optical VTA stimulation in ChR2+ mice during continuous, steady-state general anesthesia (CSSGA) with isoflurane produced behavioral and EEG evidence of arousal and restored the righting reflex in 6/6 mice. Pretreatment with the D1 receptor antagonist SCH-23390 before optical VTA stimulation inhibited the arousal responses and restoration of righting in 6/6 ChR2+ mice. In control DAT-cre mice, the VTA was targeted with a viral vector lacking the ChR2 gene (ChR2- group; n = 5). VTA optical stimulation in ChR2- mice did not restore righting or produce EEG changes during isoflurane CSSGA in 5/5 mice. These results provide compelling evidence that selective stimulation of VTA DA neurons is sufficient to induce the transition from an anesthetized, unconscious state to an awake state, suggesting critical involvement in behavioral arousal.
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The rostral ventromedial medulla (RVM) has a well-documented role in pain modulation and exerts antinociceptive and pronociceptive influences mediated by 2 distinct classes of neurons, OFF-cells and ON-cells. OFF-cells are defined by a sudden pause in firing in response to nociceptive inputs, whereas ON-cells are characterized by a "burst" of activity. Although these reflex-related changes in ON- and OFF-cell firing are critical to their pain-modulating function, the pathways mediating these responses have not been identified. The present experiments were designed to test the hypothesis that nociceptive input to the RVM is relayed through the parabrachial complex (PB). In electrophysiological studies, ON- and OFF-cells were recorded in the RVM of lightly anesthetized male rats before and after an infusion of lidocaine or muscimol into PB. The ON-cell burst and OFF-cell pause evoked by noxious heat or mechanical probing were substantially attenuated by inactivation of the lateral, but not medial, parabrachial area. Retrograde tracing studies showed that neurons projecting to the RVM were scattered throughout PB. Few of these neurons expressed calcitonin gene-related peptide, suggesting that the RVM projection from PB is distinct from that to the amygdala. These data show that a substantial component of "bottom-up" nociceptive drive to RVM pain-modulating neurons is relayed through the PB. While the PB is well known as an important relay for ascending nociceptive information, its functional connection with the RVM allows the spinoparabrachial pathway to access descending control systems as part of a recurrent circuit.
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The mesencephalic (or midbrain) locomotor region (MLR) was first described in 1966 by Shik and colleagues, who demonstrated that electrical stimulation of this region induced locomotion in decerebrate (intercollicular transection) cats. The pedunculopontine tegmental nucleus (PPT) cholinergic neurons and midbrain extrapyramidal area (MEA) have been suggested to form the neuroanatomical basis for the MLR, but direct evidence for the role of these structures in locomotor behavior has been lacking. Here, we tested the hypothesis that the MLR is composed of non-cholinergic spinally projecting cells in the lateral pontine tegmentum. Our results showed that putative MLR neurons medial to the PPT and MEA in rats were non-cholinergic, glutamatergic, and express the orexin (hypocretin) type 2 receptors. Fos mapping correlated with motor behaviors revealed that the dorsal and ventral MLR are activated, respectively, in association with locomotion and an erect posture. Consistent with these findings, chemical stimulation of the dorsal MLR produced locomotion, whereas stimulation of the ventral MLR caused standing. Lesions of the MLR (dorsal and ventral regions together) resulted in cataplexy and episodic immobility of gait. Finally, trans-neuronal tracing with pseudorabies virus demonstrated disynaptic input to the MLR from the substantia nigra via the MEA. These findings offer a new perspective on the neuroanatomic basis of the MLR, and suggest that MLR dysfunction may contribute to the postural and gait abnormalities in Parkinsonism.
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The mechanism of loss of consciousness (LOC) under anesthesia is unknown. Because consciousness depends on activity in the cortico-thalamic network, anesthetic actions on this network are likely critical for LOC. Competing theories stress the importance of anesthetic actions on bottom-up "core" thalamo-cortical (TC) vs. top-down cortico-cortical (CC) and matrix TC connections. We tested these models using laminar recordings in rat auditory cortex in vivo and murine brain slices. We selectively activated bottom-up vs. top-down afferent pathways using sensory stimuli in vivo and electrical stimulation in brain slices, and compared effects of isoflurane on responses evoked via the two pathways. Auditory stimuli in vivo and core TC afferent stimulation in brain slices evoked short latency current sinks in middle layers, consistent with activation of core TC afferents. By contrast, visual stimuli in vivo and stimulation of CC and matrix TC afferents in brain slices evoked responses mainly in superficial and deep layers, consistent with projection patterns of top-down afferents that carry visual information to auditory cortex. Responses to auditory stimuli in vivo and core TC afferents in brain slices were significantly less affected by isoflurane compared to responses triggered by visual stimuli in vivo and CC/matrix TC afferents in slices. At a just-hypnotic dose in vivo, auditory responses were enhanced by isoflurane, whereas visual responses were dramatically reduced. At a comparable concentration in slices, isoflurane suppressed both core TC and CC/matrix TC responses, but the effect on the latter responses was far greater than on core TC responses, indicating that at least part of the differential effects observed in vivo were due to local actions of isoflurane in auditory cortex. These data support a model in which disruption of top-down connectivity contributes to anesthesia-induced LOC, and have implications for understanding the neural basis of consciousness.
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How general anesthetics cause loss of consciousness is unknown. Some evidence points toward effects on the neocortex causing “top-down” inhibition, whereas other findings suggest that these drugs act via subcortical mechanisms, possibly selectively stimulating networks promoting natural sleep. To determine whether some neuronal circuits are affected before others, we used Morlet wavelet analysis to obtain high temporal resolution in the time-varying power spectra of local field potentials recorded simultaneously in discrete brain regions at natural sleep onset and during anesthetic-induced loss of righting reflex in rats. Although we observed changes in the local field potentials that were anesthetic-specific, there were some common changes in high-frequency (20–40 Hz) oscillations (reductions in frequency and increases in power) that could be detected at, or before, sleep onset and anesthetic-induced loss of righting reflex. For propofol and natural sleep, these changes occur first in the thalamus before changes could be detected in the neocortex. With dexmedetomidine, the changes occurred simultaneously in the thalamus and neocortex. In addition, the phase relationships between the low-frequency (1–4 Hz) oscillations in thalamic nuclei and neocortical areas are essentially the same for natural sleep and following dexmedetomidine administration, but a sudden change in phase, attributable to an effect in the central medial thalamus, occurs at the point of dexmedetomidine loss of righting reflex. Our data are consistent with the central medial thalamus acting as a key hub through which general anesthesia and natural sleep are initiated.
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Work in animals and humans has suggested the existence of a slow wave sleep (SWS)-promoting/electroencephalogram (EEG)-synchronizing center in the mammalian lower brainstem. Although sleep-active GABAergic neurons in the medullary parafacial zone (PZ) are needed for normal SWS, it remains unclear whether these neurons can initiate and maintain SWS or EEG slow-wave activity (SWA) in behaving mice. We used genetically targeted activation and optogenetically based mapping to examine the downstream circuitry engaged by SWS-promoting PZ neurons, and we found that this circuit uniquely and potently initiated SWS and EEG SWA, regardless of the time of day. PZ neurons monosynaptically innervated and released synaptic GABA onto parabrachial neurons, which in turn projected to and released synaptic glutamate onto cortically projecting neurons of the magnocellular basal forebrain; thus, there is a circuit substrate through which GABAergic PZ neurons can potently trigger SWS and modulate the cortical EEG.
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The activity of histaminergic neurons in the tuberomammillary nucleus (TMN) of the hypothalamus correlates with an animal's behavioral state and maintains arousal. We examined how GABAergic inputs onto histaminergic neurons regulate this behavior. A prominent hypothesis, the "flip-flop" model, predicts that increased and sustained GABAergic drive onto these cells promotes sleep. Similarly, because of the histaminergic neurons' key hub-like place in the arousal circuitry, it has also been suggested that anesthetics such as propofol induce loss of consciousness by acting primarily at histaminergic neurons. We tested both these hypotheses in mice by genetically removing ionotropic GABA(A) or metabotropic GABA(B) receptors from histidine decarboxylase-expressing neurons. At the cellular level, histaminergic neurons deficient in synaptic GABA(A) receptors were significantly more excitable and were insensitive to the anesthetic propofol. At the behavioral level, EEG profiles were recorded in nontethered mice over 24 h. Surprisingly, GABAergic transmission onto histaminergic neurons had no effect in regulating the natural sleep-wake cycle and, in the case of GABA(A) receptors, for propofol-induced loss of righting reflex. The latter finding makes it unlikely that the histaminergic TMN has a central role in anesthesia. GABA(B) receptors on histaminergic neurons were dispensable for all behaviors examined. Synaptic inhibition of histaminergic cells by GABA(A) receptors, however, was essential for habituation to a novel environment.
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J. Neurochem. (2011) 118, 571–580. The oral part of the pontine reticular formation (PnO) is a component of the ascending reticular activating system and plays a role in the regulation of sleep and wakefulness. The PnO receives glutamatergic and GABAergic projections from many brain regions that regulate behavioral state. Indirect, pharmacological evidence has suggested that glutamatergic and GABAergic signaling within the PnO alters traits that characterize wakefulness and sleep. No previous studies have simultaneously measured endogenous glutamate and GABA from rat PnO in relation to sleep and wakefulness. The present study utilized in vivo microdialysis coupled on-line to capillary electrophoresis with laser-induced fluorescence to test the hypothesis that concentrations of glutamate and GABA in the PnO vary across the sleep/wake cycle. Concentrations of glutamate and GABA were significantly higher during wakefulness than during non-rapid eye movement sleep and rapid eye movement sleep. Regression analysis revealed that decreases in glutamate and GABA accounted for a significant portion of the variance in the duration of non-rapid eye movement sleep and rapid eye movement sleep episodes. These data provide novel support for the hypothesis that endogenous glutamate and GABA in the PnO contribute to the regulation of sleep duration.
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In the United States, nearly 60,000 patients per day receive general anesthesia for surgery.1 General anesthesia is a drug-induced, reversible condition that includes specific behavioral and physiological traits — unconsciousness, amnesia, analgesia, and akinesia — with concomitant stability of the autonomic, cardiovascular, respiratory, and thermoregulatory systems.2 General anesthesia produces distinct patterns on the electroencephalogram (EEG), the most common of which is a progressive increase in low-frequency, high-amplitude activity as the level of general anesthesia deepens3,4 (Figure 1Figure 1Electroencephalographic (EEG) Patterns during the Awake State, General Anesthesia, and Sleep.). How anesthetic drugs induce and maintain the behavioral states of general anesthesia is an important question in medicine and neuroscience.6 Substantial insights can be gained by considering the relationship of general anesthesia to sleep and to coma. Humans spend approximately one third of their lives asleep. Sleep, a state of decreased arousal that is actively generated by nuclei in the hypothalamus, brain stem, and basal forebrain, is crucial for the maintenance of health.7,8 Normal human sleep cycles between two states — rapid-eye-movement (REM) sleep and non-REM sleep — at approximately 90-minute intervals. REM sleep is characterized by rapid eye movements, dreaming, irregularities of respiration and heart rate, penile and clitoral erection, and airway and skeletal-muscle hypotonia.7 In REM sleep, the EEG shows active high-frequency, low-amplitude rhythms (Figure 1). Non-REM sleep has three distinct EEG stages, with higher-amplitude, lower-frequency rhythms accompanied by waxing and waning muscle tone, decreased body temperature, and decreased heart rate. Coma is a state of profound unresponsiveness, usually the result of a severe brain injury.9 Comatose patients typically lie with eyes closed and cannot be roused to respond appropriately to vigorous stimulation. A comatose patient may grimace, move limbs, and have stereotypical withdrawal responses to painful stimuli yet make no localizing responses or discrete defensive movements. As the coma deepens, the patient's responsiveness even to painful stimuli may diminish or disappear. Although the patterns of EEG activity observed in comatose patients depend on the extent of the brain injury, they frequently resemble the high–amplitude, low-frequency activity seen in patients under general anesthesia10 (Figure 1). General anesthesia is, in fact, a reversible drug-induced coma. Nevertheless, anesthesiologists refer to it as “sleep” to avoid disquieting patients. Unfortunately, anesthesiologists also use the word “sleep” in technical descriptions to refer to unconsciousness induced by anesthetic drugs.11 (For a glossary of terms commonly used in the field of anesthesiology, see the Supplementary Appendix, available with the full text of this article at NEJM.org.) This review discusses the clinical and neurophysiological features of general anesthesia and their relationships to sleep and coma, focusing on the neural mechanisms of unconsciousness induced by selected intravenous anesthetic drugs
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The recent discovery of a barbiturate-sensitive "general anesthesia switch" mechanism localized in the rat brain stem mesopontine tegmental anesthesia area (MPTA) has challenged the current view of the nonspecific actions of general anesthetic agents in the CNS. In this study we provide electrophysiological evidence that the antinociception, which accompanies the behavioral state resembling general anesthesia following pentobarbital (PB) microinjections into the MPTA of awake rats, could be accompanied by the attenuation of sensory transmission through the spinothalamic tract (STT). Following bilateral microinjections of PB into the MPTA spontaneous firing rate (SFR), antidromic firing index (FI), and sciatic (Sc) as well as sural (Su) nerve-evoked responses (ER) of identified lumbar STT neurons in the isoflurane-anesthetized rat were quantified using extracellular recording techniques. Microinjections of PB into the MPTA significantly suppressed the SFR (47%), magnitudes of Sc- (26%) and Su-ER (36%), and FI (41%) of STT neurons. Microinjections of PB-free vehicle control did not alter any of the above-cited electrophysiological parameters. The results from this study suggest that antinociception, which occurs during the anesthesia-like state following PB microinjections into the MPTA, may be due, in part, to (in)direct inhibition of STT neurons via switching mechanism(s) located in the MPTA. This study provides a provenance for investigating electrophysiologically the actions on STT neurons of other current agents used clinically to maintain the state of general anesthesia.
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Midbrain dopamine neurons play central roles in reward processing. It is widely assumed that all dopamine neurons encode the same information. Some evidence, however, suggests functional differences between subgroups of dopamine neurons, particularly with respect to processing nonrewarding, aversive stimuli. To directly test this possibility, we recorded from and juxtacellularly labeled individual ventral tegmental area (VTA) dopamine neurons in anesthetized rats so that we could link precise anatomical position and neurochemical identity with coding for noxious stimuli. Here, we show that dopamine neurons in the dorsal VTA are inhibited by noxious footshocks, consistent with their role in reward processing. In contrast, we find that dopamine neurons in the ventral VTA are phasically excited by footshocks. This observation can explain a number of previously confusing findings that suggested a role for dopamine in processing both rewarding and aversive events. Taken together, our results indicate that there are 2 functionally and anatomically distinct VTA dopamine systems.
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Rapid eye movement (REM) sleep is a distinct brain state characterized by activated electroencephalogram and complete skeletal muscle paralysis, and is associated with vivid dreams. Transection studies by Jouvet first demonstrated that the brainstem is both necessary and sufficient for REM sleep generation, and the neural circuits in the pons have since been studied extensively. The medulla also contains neurons that are active during REM sleep, but whether they play a causal role in REM sleep generation remains unclear. Here we show that a GABAergic (γ-aminobutyric-acid-releasing) pathway originating from the ventral medulla powerfully promotes REM sleep in mice. Optogenetic activation of ventral medulla GABAergic neurons rapidly and reliably initiated REM sleep episodes and prolonged their durations, whereas inactivating these neurons had the opposite effects. Optrode recordings from channelrhodopsin-2-tagged ventral medulla GABAergic neurons showed that they were most active during REM sleep (REMmax), and during wakefulness they were preferentially active during eating and grooming. Furthermore, dual retrograde tracing showed that the rostral projections to the pons and midbrain and caudal projections to the spinal cord originate from separate ventral medulla neuron populations. Activating the rostral GABAergic projections was sufficient for both the induction and maintenance of REM sleep, which are probably mediated in part by inhibition of REM-suppressing GABAergic neurons in the ventrolateral periaqueductal grey. These results identify a key component of the pontomedullary network controlling REM sleep. The capability to induce REM sleep on command may offer a powerful tool for investigating its functions.
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Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol(®)) and 4-PIOL.
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Women recover faster from propofol anaesthesia and have been described to have a higher incidence of awareness during surgery, compared to men – an effect that may be inherent in sex differences in propofol metabolism. In an observational study, 98 ASA I-II patients treated with continuous propofol infusion were recruited. The associations between sex and CYP2B6 and UGT1A9 polymorphisms with dose- and weight-adjusted area under the total plasma level time curves (AUC) for propofol, and its metabolites propofol glucuronide (PG), 4-hydroxypropofol (OHP) and hydroxyl glucuronide metabolites 4-hydroxypropofol-1-O-β-D-glucuronide (Q1G) and 4-hydroxypropofol-4-O-β-D-glucuronide (Q4G), were analysed. Significantly higher AUC of PG (1.3 times, p = 0.03), Q1G (2.9 times, p < 0.001), Q4G (2.4 times, p < 0.01) and OHP (4.6 times, p = 0.01) were found in women (n = 53) than in men (n = 45) after intravenous infusion of propofol using target-controlled infusion system. There was, however, no significant impact of gene polymorphisms on propofol biotransformation. The results, which are supported by a previous pilot study using a propofol bolus dose, suggest that, compared to men, more rapid propofol metabolism may occur in women – a factor that may contribute to the mentioned differences in the efficacy of propofol anaesthesia between male and female patients.
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Anesthetics have been used in clinical practice for over a hundred years, yet their mechanisms of action remain poorly understood. One tempting hypothesis to explain their hypnotic properties posits that anesthetics exert a component of their effects by "hijacking" the endogenous arousal circuitry of the brain. Modulation of activity within sleep- and wake-related neuroanatomic systems could thus explain some of the varied effects produced by anesthetics. There has been a recent explosion of research into the neuroanatomic substrates affected by various anesthetics. In this review, we will highlight the relevant sleep architecture and systems and focus on studies over the past few years that implicate these sleep-related structures as targets of anesthetics. These studies highlight a promising area of investigation regarding the mechanisms of action of anesthetics and provide an important model for future study.
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The "ascending reticular activating system" theory proposed that neurons in the upper brainstem reticular formation projected to forebrain targets that promoted wakefulness. More recent formulations have emphasized that most neurons at the pontomesencephalic junction that participate in these pathways are actually in monoaminergic and cholinergic cell groups. However, cell-specific lesions of these cell groups have never been able to reproduce the deep coma seen after acute paramedian midbrain lesions that transect ascending axons at the caudal midbrain level. To determine whether the cortical afferents from the thalamus or the basal forebrain were more important in maintaining arousal, we first placed large cell-body-specific lesions in these targets. Surprisingly, extensive thalamic lesions had little effect on electroencephalographic (EEG) or behavioral measures of wakefulness or on c-Fos expression by cortical neurons during wakefulness. In contrast, animals with large basal forebrain lesions were behaviorally unresponsive and had a monotonous sub-1-Hz EEG, and little cortical c-Fos expression during continuous gentle handling. We then retrogradely labeled inputs to the basal forebrain from the upper brainstem, and found a substantial input from glutamatergic neurons in the parabrachial nucleus and adjacent precoeruleus area. Cell-specific lesions of the parabrachial-precoeruleus complex produced behavioral unresponsiveness, a monotonous sub-1-Hz cortical EEG, and loss of cortical c-Fos expression during gentle handling. These experiments indicate that in rats the reticulo-thalamo-cortical pathway may play a very limited role in behavioral or electrocortical arousal, whereas the projection from the parabrachial nucleus and precoeruleus region, relayed by the basal forebrain to the cerebral cortex, may be critical for this process.
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Microinjection of pentobarbital into a restricted region of rat brainstem, the mesopontine tegmental anesthesia area (MPTA), induces a reversible anesthesia-like state characterized by loss of the righting reflex, atonia, antinociception, and loss of consciousness as assessed by electroencephalogram synchronization. We examined cerebral activity during this state using FOS expression as a marker. Animals were anesthetized for 50 min with a series of intracerebral microinjections of pentobarbital or with systemic pentobarbital and intracerebral microinjections of vehicle. FOS expression was compared with that in awake animals microinjected with vehicle. Neural activity was suppressed throughout the cortex whether anesthesia was induced by systemic or MPTA routes. Changes were less consistent subcortically. In the zona incerta and the nucleus raphe pallidus, expression was strongly suppressed during systemic anesthesia, but only mildly during MPTA-induced anesthesia. Dissociation was seen in the tuberomammillary nucleus where suppression occurred during systemic-induced anesthesia only, and in the lateral habenular nucleus where activity was markedly increased during systemic-induced anesthesia but not following intracerebral microinjection. Several subcortical nuclei previously associated with cerebral arousal were not affected. In the MPTA itself FOS expression was suppressed during systemic anesthesia. Differences in the pattern of brain activity in the two modes of anesthesia are consistent with the possibility that anesthetic endpoints might be achieved by alternative mechanisms: direct drug action for systemic anesthesia or via ascending pathways for MPTA-induced anesthesia. However, it is also possible that systemically administered agents induce anesthesia, at least in part, by a primary action in the MPTA with cortical inhibition occurring secondarily.
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A morphological analysis was undertaken of both the dispersion characteristics and tissue content of dopamine (DA) microinjected acutely into the brain-stem of the anesthetized rat. 14C-DA, with a specific activity of 56-62 mCi/mMol, was infused unilaterally into the pars compacta of the substantia nigra in one of four test volumes: 0.5, 1.0, 4.0 or 8.0 microliters. The concentration of the 14C-DA solution was 1.0 microCi/microliter, equivalent to 3.01 micrograms/microliters, which was delivered at an injection rate of 1.0 microliter per 45 sec. At an interval of either one min or 15 min following the microinjection, the rat's brain was removed rapidly from its calvarium, flash frozen and then cut in the coronal plane on a freezing microtome in 500 micron slabs. After each of the respective serial slabs was mounted on glass, the Eik Nes-Brizzee trochar technique for the discrete removal of tissue samples was used to obtain 0.5 mm dia. cylindrical plugs of meso-diencephalic tissue at distances from the site of injection ranging from 0.5 to 2.5 mm, center to center. Each sample plug was subsequently solubilized and 14C-DA activity quantitated by liquid scintillation spectrometry. The results show that regardless of volume, the spatial patterning of the microinjected solution assumes a tear-drop or pear shape, not a sphere. Further, as the volume of the injection is increased from 0.5 to 8.0 microliters, the magnitude of the dispersion of 14C-DA is enhanced throughout the surrounding parenchyma, but not in a linear fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
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Concussion, asphyxia, and systemically administered general anesthetics all induce reversible depression of the organism's response to noxious stimuli as one of the elements of loss of consciousness. This is so even for barbiturate anesthetics, which have only modest analgesic efficacy at subanesthetic doses. Little is known about the neural circuits involved in this form of antinociception, although for anesthetic agents, at least, it is usually presumed that the drugs act in widely distributed regions of the nervous system. We now report the discovery of a focal zone in the brainstem mesopontine tegmentum in rats at which microinjection of minute quantities of pentobarbital induces a transient, reversible anesthetic-like state with non-responsiveness to noxious stimuli, flaccid atonia, and absence of the righting reflex. The behavioral suppression is accompanied by slow-wave EEG and, presumably, loss of consciousness. This zone, which we refer to as the mesopontine tegmental anesthesia locus (MPTA), apparently contains a barbiturate-sensitive 'switch' for both cortical and spinal activity. The very existence of the MPTA locus has implications for an understanding of the neural circuits that control motor functions and pain sensation, and for the cerebral representation of consciousness.
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Classical anesthetics of the gamma-aminobutyric acid type A receptor (GABA(A))-enhancing class (e.g., pentobarbital, chloral hydrate, muscimol, and ethanol) produce analgesia and unconsciousness (sedation). Dissociative anesthetics that antagonize the N-methyl-D-aspartate (NMDA) receptor (e.g., ketamine, MK-801, dextromethorphan, and phencyclidine) produce analgesia but do not induce complete loss of consciousness. To understand the mechanisms underlying loss of consciousness and analgesia induced by general anesthetics, we examined the patterns of expression of c-Fos protein in the brain and correlated these with physiological effects of systemically administering GABAergic agents and ketamine at dosages used clinically for anesthesia in rats. We found that GABAergic agents produced predominantly delta activity in the electroencephalogram (EEG) and sedation. In contrast, anesthetic doses of ketamine induced sedation, followed by active arousal behaviors, and produced a faster EEG in the theta range. Consistent with its behavioral effects, ketamine induced Fos expression in cholinergic, monoaminergic, and orexinergic arousal systems and completely suppressed Fos immunoreactivity in the sleep-promoting ventrolateral preoptic nucleus (VLPO). In contrast, GABAergic agents suppressed Fos in the same arousal-promoting systems but increased the number of Fos-immunoreactive neurons in the VLPO compared with waking control animals. All anesthetics tested induced Fos in the spinally projecting noradrenergic A5-7 groups. 6-hydroxydopamine lesions of the A5-7 groups or ibotenic acid lesions of the ventrolateral periaqueductal gray matter (vlPAG) attenuated antinociceptive responses to noxious thermal stimulation (tail-flick test) by both types of anesthetics. We hypothesize that neural substrates of sleep-wake behavior are engaged by low-dose sedative anesthetics and that the mesopontine descending noradrenergic cell groups contribute to the analgesic effects of both NMDA receptor antagonists and GABA(A) receptor-enhancing anesthetics.
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The mechanisms through which general anaesthetics, an extremely diverse group of drugs, cause reversible loss of consciousness have been a long-standing mystery. Gradually, a relatively small number of important molecular targets have emerged, and how these drugs act at the molecular level is becoming clearer. Finding the link between these molecular studies and anaesthetic-induced loss of consciousness presents an enormous challenge, but comparisons with the features of natural sleep are helping us to understand how these drugs work and the neuronal pathways that they affect. Recent work suggests that the thalamus and the neuronal networks that regulate its activity are the key to understanding how anaesthetics cause loss of consciousness.
CrossRef Myers RD, Hoch DB (1978) 14C-dopamine microinjected into the brainstem of the rats: dispersion kinetics, site content and functional dose
  • M Morales
  • E B Margolis
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