Article

Patterns of Neural Activity in the Mouse Brain: Wakefulness vs. General Anesthesia

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Abstract

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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... used [15,16]. To the best of our knowledge, except for c-fos, very few other IEG products have been used to detect neural activity to determine the neural basis of general anesthesia or sleep-arousal [17][18][19]. Therefore, in this review, we focused on studies that investigated the neural basis of mechanisms of action of general anesthetics based on a c-fos expression. ...
... 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. ...
... In addition to the subcortical brain nucleus, it is widely known that the neural activity of the cerebral cortex itself also plays an important role in the general anesthetic induced-unconsciousness. Previous studies indicated that systemic administration of pentobarbital, a classical GABAergic sedative, caused widespread suppression of c-fos expression in the cerebral cortex of both mice and rats [19,23,64,87], suggesting the activity of cerebral cortex was suppressed in the anesthetic-induced unconsciousness. However, the precise mechanism of how general anesthetics interfere with activity and/or connectivity of the cerebral cortex to induce unconsciousness is not well known. ...
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.
... Since these early investigations, the hypothalamus has been increasingly recognized as a loose confederation of autonomous neurons that regulate many essential social and homeostatic functions (Sternson, 2013;Wu et al., 2014;Scott et al., 2015;Tan et al., 2016;Allen et al., 2017;Leib et al., 2017), including sleep and wake . More specifically, the preoptic area of the hypothalamus (POA) is known to modulate arousal in both natural (sleep and wake) (Gallopin et al., 2000;Lu et al., 2000Lu et al., , 2002McGinty and Szymusiak, 2001;Gong et al., 2004;Chung et al., 2017) as well as drug-induced (anestheticinduced unconsciousness) states (Nelson et al., 2002;Lu et al., 2008;Li et al., 2009;Moore et al., 2012;Liu et al., 2013;Han et al., 2014;McCarren et al., 2014;Zhang Y. et al., 2015;Yatziv et al., 2020). However, the degree to which the same population of neurons within the POA modulates arousal in both sleep and anesthesia is unclear. ...
... These findings have led to what is known as the "shared circuitry hypothesis" of anesthesia, which posits that anesthetics exert their hypnotic effects in part by acting on the neural circuitry that regulates endogenous sleep and wake. More specifically, this theory hypothesizes that anesthetics cause unconsciousness via activation of sleeppromoting populations and/or inhibition of wake-promoting populations, rather than by the wet-blanket theory of nonspecific, global disruption of CNS function (Lydic and Biebuyck, 1994;Yatziv et al., 2020). ...
... Although a number of studies have implicated sleep-and wake-regulating brain areas in anesthetic hypnosis, controversy remains as to whether the neural circuits, and more specifically, the same neurons shaping sleep and wakefulness actually do influence the anesthetic state in vivo. Past work has demonstrated that the POA, in addition to modulating sleep and wake, is also capable of modulating anesthetic-induced unconsciousness (Nelson et al., 2002;Lu et al., 2008;Li et al., 2009;Moore et al., 2012;Liu et al., 2013;Han et al., 2014;McCarren et al., 2014;Zhang Y. et al., 2015;Yatziv et al., 2020). However, the degree to which the same population of neurons within the POA modulates arousal in both sleep and anesthesia is unclear. ...
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The role of the hypothalamic preoptic area (POA) in arousal state regulation has been studied since Constantin von Economo first recognized its importance in the early twentieth century. Over the intervening decades, the POA has been shown to modulate arousal in both natural (sleep and wake) as well as drug-induced (anesthetic-induced unconsciousness) states. While the POA is well known for its role in sleep promotion, populations of wake-promoting neurons within the region have also been identified. However, the complexity and molecular heterogeneity of the POA has made distinguishing these two populations difficult. Though multiple lines of evidence demonstrate that general anesthetics modulate the activity of the POA, the region’s heterogeneity has also made it challenging to determine whether the same neurons involved in sleep/wake regulation also modulate arousal in response to general anesthetics. While a number of studies show that sleep-promoting POA neurons are activated by various anesthetics, recent work suggests this is not universal to all arousal-regulating POA neurons. Technical innovations are making it increasingly possible to classify and distinguish the molecular identities of neurons involved in sleep/wake regulation as well as anesthetic-induced unconsciousness. Here, we review the current understanding of the POA’s role in arousal state regulation of both natural and drug-induced forms of unconsciousness, including its molecular organization and connectivity to other known sleep and wake promoting regions. Further insights into the molecular identities and connectivity of arousal-regulating POA neurons will be critical in fully understanding how this complex region regulates arousal states.
... Several studies have confirmed that anesthetics impacted neural activities, including significant downregulation of Cxcl12 mRNA expression in primary hippocampal neurons [19], suppressing neural activities in cerebral cortex [20], inhibiting excitatory transmitter release by blocking presynaptic Ca 2+ channels or extracellular mechanisms [21], and leading to distinct spatiotemporal activity in principal neurons of the mouse olfactory cortex [22]. Not only that, a preliminary study also showed that the modulation of functional connectivity by low-frequency rTMS varied considerably among the isoflurane, propofol and dexmedetomidine groups [23]. ...
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How general anesthesia (GA) induces loss of consciousness remains unclear, and whether diverse anesthetic drugs and sleep share a common neural pathway is unknown. Previous studies have revealed that many GA drugs inhibit neural activity through targeting GABA receptors. Here, using Fos staining, ex vivo brain slice recording, and in vivo multi-channel electrophysiology, we discovered a core ensemble of hypothalamic neurons in and near the supraoptic nucleus, consisting primarily of neuroendocrine cells, which are persistently and commonly activated by multiple classes of GA drugs. Remarkably, chemogenetic or brief optogenetic activations of these anesthesia-activated neurons (AANs) strongly promote slow-wave sleep and potentiates GA, whereas conditional ablation or inhibition of AANs led to diminished slow-wave oscillation, significant loss of sleep, and shortened durations of GA. These findings identify a common neural substrate underlying diverse GA drugs and natural sleep and reveal a crucial role of the neuroendocrine system in regulating global brain states. VIDEO ABSTRACT.
Article
SIM1-expressing paraventricular hypothalamus (PVH) neurons are key regulators of energy balance. Within the PVH SIM1 population, melanocortin-4 receptor-expressing (PVH MC4R ) neurons are known to regulate satiety and bodyweight, yet they account for only half of PVH SIM1 neuron-mediated regulation. Here we report that PVH prodynorphin-expressing (PVH PDYN ) neurons, which notably lack MC4Rs, function independently and additively with PVH MC4R neurons to account for the totality of PVH SIM1 neuron-mediated satiety. Moreover, PVH PDYN neurons are necessary for prevention of obesity in an independent but equipotent manner to PVH MC4R neurons. While PVH PDYN and PVH MC4R neurons both project to the parabrachial complex (PB), they synaptically engage distinct efferent nodes, the pre-locus coeruleus (pLC), and central lateral parabrachial nucleus (cLPBN), respectively. PB-projecting PVH PDYN neurons, like PVH MC4R neurons, receive input from interoceptive ARC AgRP neurons, respond to caloric state, and are sufficient and necessary to control food intake. This expands the CNS satiety circuitry to include two non-overlapping PVH to hindbrain circuits. Li et al. discover that PVH PDYN neurons, additively with PVH MC4R neurons, are responsible for PVH-mediated satiety. Both PVH neurons are distinct, are equipotent, and use parallel projections to different aspects of the parabrachial complex to cause satiety and prevent obesity.
Article
The MPTA (mesopontine tegmental anesthesia area) is a key node in a network of axonal pathways that collectively engage the key components of general anesthesia: immobility and atonia, analgesia, amnesia and loss-of-consciousness. In this study we have applied double retrograde tracing to analyze MPTA connectivity, with a focus on axon collateralization. Prior tracer studies have shown that collectively, MPTA neurons send descending projections to spinal and medullary brain targets associated with atonia and analgesia as well as ascending projections to forebrain structures associated with amnesia and arousal. Here we ask whether individual MPTA neurons collateralize broadly as might be expected of modulatory circuitry, sending axonal branches to both caudal and to rostral targets, or whether connectivity is more selective. Two distinguishable retrograde tracers were microinjected into pairs ("dyads") of known synaptic targets of the MPTA, one caudal and one rostral. We found that neurons that were double-labeled, and hence project to both targets were rare, constituting <0.5% on average of all MPTA neurons that project to these targets. The large majority sent axons either caudally, presumably to mediate mobility and/or antinociception, or rostrally, presumably to mediate mnemonic and/or arousal/cognitive functions. MPTA neurons with descending vs. ascending projections also differed in size and shape, supporting the conclusion that they constitute distinct neuronal populations. From these and prior observations we conclude that the MPTA has a hybrid architecture including neurons with heterogeneous patterns of connectivity, some highly collateralized and some more targeted.
Article
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.
Article
Intensive Care Unit (ICU) or emergency care patients, exposed to traumatic events, are at increased risk for Post-Traumatic Stress Disorder (PTSD) development. Commonly used sedative/anesthetic agents can interfere with the mechanisms of memory formation, exacerbating or attenuating the memory for the traumatic event, and subsequently promote or reduce the risk of PTSD development. Here, we evaluated the effects of ketamine, dexmedetomidine and propofol on fear memory consolidation and subsequent cognitive and emotional alterations related to traumatic stress exposure. Immediately following an inhibitory avoidance training, rats were intraperitoneally injected with ketamine (100–125 mg/kg), dexmedetomidine (0.3-0.4 mg/kg) or their vehicle and tested for 48 h memory retention. Furthermore, the effects of ketamine (125 mg/kg), dexmedetomidine (0.4 mg/kg), propofol (300 mg/kg) or their vehicle on long-term memory and social interaction were evaluated two weeks after drug injection in a rat PTSD model. Ketamine anesthesia increased memory retention without altering the traumatic memory strength in the PTSD model. However, ketamine induced a long-term reduction of social behavior. Conversely, dexmedetomidine markedly impaired memory retention, without affecting long-lasting cognitive or emotional behaviors in the PTSD model. We have previously shown that propofol anesthesia enhanced 48 h memory retention. Here, we found that propofol induced an enduring traumatic memory enhancement and anxiogenic effects in the PTSD model. These findings provide new evidence for clinical studies showing that the use of ketamine or propofol anesthesia in emergency care and ICU might be more likely to promote the development of PTSD, while dexmedetomidine might have prophylactic effects.
Article
We review evidence that the induction of anesthesia with GABAergic agents is mediated by a network of dedicated axonal pathways, which convey a suppressive signal to remote parts of the central nervous system. The putative signal originates in an anesthetic-sensitive locus in the brainstem that we refer to as the mesopontine tegmental anesthesia area (MPTA). This architecture stands in contrast to the classical notion that anesthetic molecules themselves directly mediate anesthetic induction after global distribution by the vascular circulation. The MPTA came to light in a systematic survey of the rat brain as a singular locus at which microinjection of minute quantities of GABAergic anesthetics is able to reversibly induce a state resembling surgical anesthesia. The rapid onset of anesthesia, the observed target specificity, and the fact that effective doses are far too small to survive dilution during vascular redistribution to distant areas in the central nervous system are all incompatible with the classical global suppression model. Lesioning the MPTA selectively reduces the animal's sensitivity to systemically administered anesthetics. Taken together, the microinjection data show that it is sufficient to deliver γ-aminobutyric acid A receptor (GABAA-R) agonists to the MPTA to induce an anesthesia-like state and the lesion data indicate that MPTA neurons are necessary for anesthetic induction by the systemic route at clinically relevant doses. Known connectivity of the MPTA provides a scaffold for defining the specific projection pathways that mediate each of the functional components of anesthesia. Because MPTA lesions do not induce coma, the MPTA is not a key arousal nucleus essential for maintaining the awake state. Rather, it appears be a "gatekeeper" of arousal function, a major element in a flip-flop switching mechanism that executes rapid and reversible transitions between the awake and the anesthetic state.
Article
Background: Although the effects of propofol on cerebral metabolism have been studied in animals, these effects have yet to be directly examined in humans. Consequently, we used positron emission tomography (PET) to demonstrate in vivo the regional cerebral metabolic changes that occur in humans during propofol anesthesia, Methods: Six volunteers each underwent two PET scans; one scan assessed awake-baseline metabolism, and the other assessed metabolism during anesthesia with a propofol infusion titrated to the point of unresponsiveness (mean rate +/- SD = 7.8 +/- 1.5 mg.kg(-1).h(-1)). Scans were obtained using the (18)fluorodeoxyglucose technique. Results: Awake whole-brain glucose metabolic rates (GMR) averaged 29 +/- 8 mu moles.100g(-1)min(-1) (mean +/- SD). Anesthetized whole-brain GMR averaged 13 +/- 4 mu moles.100g(-1).min(-1) (paired t test, P less than or equal to 0.007), GMR decreased in all measured areas during anesthesia. However, the decrease in GMR was not uniform. Cortical metabolism was depressed 58%, whereas subcortical metabolism was depressed 48% (P less than or equal to 0.001). Marked differences within cortical regions also occurred. In the medial and subcortical regions, the largest percent decreases occurred in the left anterior cingulate and the inferior colliculus. Conclusion: Propofol produced a global metabolic depression on the human central nervous system, The metabolic pattern evident during anesthesia was reproducible and differed from that seen in the awake condition. These findings are consistent with those from previous animal studies and suggest PET may be useful for investigating the mechanisms of anesthesia in humans.
Article
General anesthetic agents induce loss of consciousness coupled with suppression of movement, analgesia and amnesia. Although these diverse functions are mediated by neural structures located in wide-ranging parts of the neuraxis, anesthesia can be induced rapidly and reversibly by bilateral microinjection of minute quantities of GABAA -R agonists at a small, focal locus in the mesopontine tegmentum (MPTA). State switching under these circumstances is presumably executed by dedicated neural pathways and does not require widespread distribution of the anesthetic agent itself, the classical assumption regarding anesthetic induction. Here we asked whether these pathways serve each hemisphere independently, or whether there is bilateral redundancy such that the MPTA on each side is capable of anesthetizing the entire brain. Either of two GABAA -R ligands were microinjected unilaterally into the MPTA in awake rats, the barbiturate modulator pentobarbital and the direct receptor agonist muscimol. Both agents, microinjected on either side, induced clinical anesthesia including bilateral atonia, bilateral analgesia and bilateral changes in cortical activity. The latter was monitored using c-fos expression and electroencephalography. This action, however, was not simply a consequence of suppressing spike activity in MPTA neurons as unilateral (or bilateral) microinjection of the local anesthetic lidocaine at the same locus failed to induce anesthesia. We propose a model of the state-switching circuitry that accounts for the bilateral action of unilateral microinjection and also for the observation that inactivation with lidocaine is not equivalent to inhibition with GABAA -R agonists. This is a step in defining the overall switching circuitry that underlies anesthesia. This article is protected by copyright. All rights reserved.
Article
Noninvasive functional imaging holds great promise for the future of translational research, due to the ability to directly compare between preclinical and clinical models of psychiatric disorders. Despite this potential, concerns have been raised regarding the necessity to anesthetize rodent and monkey subjects during these procedures, because anesthetics may alter neuronal activity. For example, in studies of drugs of abuse and alcohol, it is not clear to what extent anesthesia can interfere with drug-induced neural activity. Therefore, the current study investigated whole brain c-Fos activation following isoflurane anesthesia as well as ethanol-induced activation of c-Fos in anesthetized mice. In the first experiment, we examined effects of 1 or 3 sessions of gaseous isoflurane on c-Fos activation across the brain in male C57BL/6J mice. Isoflurane administration led to c-Fos activation in several areas, including the piriform cortex and lateral septum. Lower or similar levels of activation in these areas were detected after three sessions of isoflurane, suggesting that multiple exposures may eliminate some of the enhanced neuronal activation caused by acute isoflurane. In the second experiment, we investigated the ability of ethanol injection (1.5 or 2.5 g/kg i.p.) to induce c-Fos activation under anesthesia. Following three sessions of isoflurane, 1.5 g/kg of ethanol induced c-Fos in the central nucleus of amygdala, piriform cortex, and centrally-projecting Edinger-Westphal nucleus. This induction was lower after 2.5 g/kg of ethanol. These results demonstrate that ethanol-induced neural activation can be detected in the presence of isoflurane anesthesia. They also suggest, that while habituation to isoflurane helps reduce neuronal activation, interaction between effects of anesthesia and alcohol can occur. Studies using fMRI imaging could benefit from using habituated animals and dose response analyses.
Article
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.
Article
Background: Despite seventeen decades of continuous clinical use, the neuronal mechanisms through which volatile anesthetics act to produce unconsciousness remain obscure. One emerging possibility is that anesthetics exert their hypnotic effects by hijacking endogenous arousal circuits. A key sleep-promoting component of this circuitry is the ventrolateral preoptic nucleus (VLPO), a hypothalamic region containing both state-independent neurons and neurons that preferentially fire during natural sleep. Results: Using c-Fos immunohistochemistry as a biomarker for antecedent neuronal activity, we show that isoflurane and halothane increase the number of active neurons in the VLPO, but only when mice are sedated or unconscious. Destroying VLPO neurons produces an acute resistance to isoflurane-induced hypnosis. Electrophysiological studies prove that the neurons depolarized by isoflurane belong to the subpopulation of VLPO neurons responsible for promoting natural sleep, whereas neighboring non-sleep-active VLPO neurons are unaffected by isoflurane. Finally, we show that this anesthetic-induced depolarization is not solely due to a presynaptic inhibition of wake-active neurons as previously hypothesized but rather is due to a direct postsynaptic effect on VLPO neurons themselves arising from the closing of a background potassium conductance. Conclusions: Cumulatively, this work demonstrates that anesthetics are capable of directly activating endogenous sleep-promoting networks and that such actions contribute to their hypnotic properties.
Article
The brain histaminergic system plays a critical role in maintenance of arousal. Previous studies suggest that histaminergic neurotransmission might be a potential mediator of general anesthetic actions. However, it is not clear whether histaminergic tuberomamillary nucleus (TMN) is necessarily involved in the sedative/hypnotic effects of general anesthetics. Male Long Evans rats underwent either TMN orexin-saporin/sham lesion or implantation of intracerebroventricular cannula 2 weeks before the experiment. The behavioral endpoint of loss of righting reflex was used to assess the hypnotic property of isoflurane, propofol, pentobarbital, and ketamine in animals. Histaminergic cell loss was assessed by adenosine deaminase expression in the TMN using immunohistochemistry. Rats with bilateral TMN orexin-saporin lesion induced an average 72% loss of histaminergic cells compared with sham-lesion rats. TMN orexin-saporin lesion or intracerebroventricular administration of triprolidine (an H1 receptor antagonist) decreased the 50% effective concentration for loss of righting reflex value and prolonged emergence time to isoflurane anesthesia. However, TMN orexin-saporin lesion had no significant effect on the anesthetic sensitivity to propofol, pentobarbital, and ketamine. These findings suggest a role of the TMN histaminergic neurons in modulating isoflurane anesthesia and that the neural circuits for isoflurane-induced hypnosis may differ from those of γ-aminobutyric acid-mediated anesthetics and ketamine.
Article
The purpose of this study was to compare effects of various anesthetic drugs on expression of Fos protein in the supraspinal neurons of rats. Optimum anesthesia was maintained under monitoring of EEG. Degree of Fos expression was evaluated by counts of Fos-immunoreactive (ir) neurons. Urethane induced the largest amount of Fos-ir neurons (n = 8439), while fentanyl/midazolam the smallest (n = 112). α-Chloralose, halothane, pentobarbital sodium and urethane/α-chloralose induced amounts in between. The results indicate that fentanyl/midazolam is the most recommendable anesthetic drug in the Fos expression study.
Article
The mechanisms underlying volatile anesthesia agents are not well elucidated. Emerging researches have focused on the participation of γ-aminobutyric acid (GABA) neurons but there still lacks morphological evidence. To elucidate the possible activation of GABAergic neurons by sevoflurane inhalation in morphology, Fos (as neuronal activity marker) and GABA neurons double labeling were observed on the brain of glutamic acid decarboxylase (GAD) 67-GFP knock-in mice after sevoflurane inhalation. Twenty GAD67-GFP knock-in mice were divided into three groups: S1 group: incomplete anesthesia state induced by sevoflurane; S2 group: complete anesthesia state induced by sevoflurane; control(C) group. Sevoflurane induced a significant increase of Fos expression in the dorsomedial hypothalamic nucleus (DM), periaqueductal grey (PAG), hippocampus (CA1, DG), paraventricular thalamic nucleus (PV), lateral septal nucleus (LS), and cingulate cortex (Cg1 and Cg2) in S1 group compared to C group, and increase of Fos expression in S2 group compared to S1 group. In S2 group, Fos was only expressed in the medial amygdaloid nucleus (MeA), Edinger-Westphal (E-W) nucleus, arcuate hypothalamic nucleus (Arc) and the ventral part of paraventricular hypothalamic nucleus (PaV). Double immunofluroscent staining indicated that in LS, almost all Fos were present in GABAergic neurons. In CA1, DG, DM, cg1, cg2, and PAG, Fos was expressed as well, but only few were present in GABAergic neurons. Fos expression was very high in thalamus, but no coexistence were found as no GABAergic neuron was detected in this area. Our results provided morphological evidence that GABAergic transmission in specific brain areas may participate in the sevoflurane-induced anesthesia.
Article
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.
Article
The expression of Fos, the protein product of the primary response gene c-fos, was used metabolically to map the short-term (1 hr) effects of urethane and sodium pentobarbital anesthesia in rat. Subsequently, urethane-anesthetized rats were used to study the integrated response to electrical stimulation (1-1.5 hr) of the pontine parabrachial nucleus (PBN), an important center for relay of autonomic information in the brain. Immunohistochemistry was used to localize Fos-like immunoreactivity (FLI) in the brain. To approximate amounts of FLI in the conscious animal, rats were killed immediately after attaining surgical anesthesia with sodium pentobarbital (50 mg/kg) or urethane (1.2-1.7 gm/kg). No FLI was found in the brains of these rats. In rats killed 1 hr after anesthesia with sodium pentobarbital, FLI was found only in the habenulae. After 1 h of urethane anesthesia, low levels of FLI were found in the following areas: nucleus of the tractus solitarius (NTS); caudal and rostral ventrolateral medulla (VLM); lateral PBN; ventromedial, paraventricular, and supraoptic nuclei (SON) of the hypothalamus; medial preoptic area; central nucleus of the amygdala (ACE); endopiriform cortex; insular cortex; piriform cortex; and islands of Calleja. Electrical stimulation of the PBN (10 sec on, 10 sec off; 15-50 microA at 20 Hz for 60-90 min) in rats anesthetized with urethane led to increases in mean arterial pressure (10-30 mm Hg) and to ipsilateral increases of FLI in the lateral PBN, dorsal division of SON, ACE, endopiriform nucleus, insular cortex, piriform cortex, and islands of Calleja. In two animals, ipsilateral increases were found in the ventromedial hypothalamus and medial amygdaloid nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product, c-fos protein, can be identified by immunohistochemical techniques. Therefore, c-fos expression might be used as a marker for neuronal activity throughout the neuraxis following peripheral stimulation. This study has analyzed patterns of c-fos expression in both control and anesthetized animals and in anesthetized rats subjected to various forms of peripheral stimulation. Labeled cells were counted in the spinal cord, brainstem, hypothalamus, and thalamus. Little c-fos immunoreactivity was found in control animals. Prolonged inhalational anesthesia increased the number of labeled cells at several brainstem sites. Noxious stimulation of anesthetized rats induced c-fos within the neuraxis in patterns consistent with data obtained from electrophysiological studies and in additional locations for which few direct electrophysiological data are available, such as the ventrolateral medulla, the posterior hypothalamic nucleus, and the reuniens and paraventricular thalamic nuclei. Gentle mechanical stimulation was ineffective in inducing c-fos-like protein. The data suggest that c-fos can be used as a transynaptic marker for neuronal activity following noxious stimulation. However, c-fos is expressed only in some kinds of neurons following peripheral stimulation, and it therefore may be an incomplete marker for nociresponsive activity. In addition, at least a few neurons express c-fos protein in the absence of noxious stimulation. Experiments analyzing c-fos expression must be designed with care, as both extraneous stimuli and anesthetic depth influence the results.
Article
The basal expression of the proto-oncogene c-fos was studied by Northern blot analysis in different regions of the rat brain during 24 h. A striking spontaneous oscillation of c-fos mRNA expression was detected in animals kept in basal conditions with a 12 h light/12 h dark cycle. In these animals c-fos mRNA was just detectable during the rest hours (morning through afternoon), and was high during the activity hours (night). The periodicity of this oscillation persisted and became free-running when the animals were exposed for 6 consecutive days to constant light or darkness. It was thus demonstrated that the fluctuation of c-fos expression is circadian and is not created by the light-dark cycle, but the latter exerts a synchronizing effect. The oscillation of c-fos mRNA was modified by manipulations of the rest-activity cycle. In particular, the fluctuation observed in basal conditions was inverted, keeping the animals awake during the rest hours (diurnal) and allowing them to sleep in the activity period (nocturnal). These data indicated a close relationship between the oscillation of c-fos expression and the rest-activity cycle. Finally, electroencephalographic (EEG) monitoring was performed under behavioural control for 3 h before the animals were killed. These experiments confirmed that, irrespective of the time of day, the EEG pattern typical of a state of sleep (including both slow waves and paradoxical sleep) was associated with low or undetectable c-fos levels, whereas the protracted EEG desynchronization corresponding to wakefulness was associated with high c-fos expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Activation of the proto-oncogene c-fos in the brain was described initially almost a decade ago and represents one of the most studied immediate early genes in the brain. Transient c-fos expression in the central nervous system was first observed after seizure activity and following noxious stimulation in the spinal cord. Since then, multiple studies have shown that different stimuli can induce c-fos expression. Seizure activity induces rapid and transient expression of c-fos in hippocampal structures. Similarly, transient activation of c-fos follows cortical brain injury in a pattern that resembles that of spreading depression. Many other stimuli have been shown to induce the expression of this proto-oncogene in the brain and c-fos immunostaining and in situ hybridization are now used to map brain metabolism under different physiological and non-physiological conditions. Here we review the variety of inducible patterns of c-fos expression in the brain.
Article
The paradigmatic assumption that REM sleep is the physiological equivalent of dreaming is in need of fundamental revision. A mounting body of evidence suggests that dreaming and REM sleep are dissociable states, and that dreaming is controlled by forebrain mechanisms. Recent neuropsychological, radiological, and pharmacological findings suggest that the cholinergic brain stem mechanisms that control the REM state can only generate the psychological phenomena of dreaming through the mediation of a second, probably dopaminergic, forebrain mechanism. The latter mechanism (and thus dreaming itself) can also be activated by a variety of nonREM triggers. Dreaming can be manipulated by dopamine agonists and antagonists with no concomitant change in REM frequency, duration, and density. Dreaming can also be induced by focal forebrain stimulation and by complex partial (forebrain) seizures during nonREM sleep, when the involvement of brainstem REM mechanisms is precluded. Likewise, dreaming is obliterated by focal lesions along a specific (probably dopaminergic) forebrain pathway, and these lesions do not have any appreciable effects on REM frequency, duration, and density. These findings suggest that the forebrain mechanism in question is the final common path to dreaming and that the brainstem oscillator that controls the REM state is just one of the many arousal triggers that can activate this forebrain mechanism. The "REM-on" mechanism (like its various NREM equivalents) therefore stands outside the dream process itself, which is mediated by an independent, forebrain "dream-on" mechanism.
Article
Little is known about how GABAergic inputs interact with excitatory inputs under conditions that maintain physiological concentrations of intracellular anions. Using extracellular and gramicidin perforated-patch recording, we show that somatic and dendritic GABA responses in mature cortical pyramidal neurons are depolarizing from rest and can facilitate action potential generation when combined with proximal excitatory input. Dendritic GABA responses were excitatory regardless of timing, whereas somatic GABA responses were inhibitory when coincident with excitatory input but excitatory at earlier times. These excitatory actions of GABA occur even though the GABA reversal potential is below action potential threshold and largely uniform across the somato-dendritic axis, and arise when GABAergic inputs are temporally or spatially isolated from concurrent excitation. Our findings demonstrate that under certain circumstances GABA will have an excitatory role in synaptic integration in the cortex.
Article
Some neurophysiologic similarities between sleep and anesthesia suggest that an anesthetized state may reverse effects of sleep deprivation. The effect of anesthesia on sleep homeostasis, however, is unknown. To test the hypothesis that recovery from sleep deprivation occurs during anesthesia, the authors followed 24 h of sleep deprivation in the rat with a 6-h period of either ad libitum sleep or propofol anesthesia, and compared subsequent sleep characteristics. With animal care committee approval, electroencephalographic/electromyographic electrodes and intrajugular cannulae were implanted in 32 rats. After a 7-day recovery and 24-h baseline electroencephalographic/electromyographic recording period, rats were sleep deprived for 24 h by the disk-over-water method. Rats then underwent 6 h of either propofol anesthesia (n = 16) or ad libitum sleep with intralipid administration (n = 16), followed by electroencephalographic/electromyographic monitoring for 72 h. In control rats, increases above baseline in non-rapid eye movement sleep, rapid eye movement sleep, and non-rapid eye movement delta power persisted for 12 h after 24 h of sleep deprivation. Recovery from sleep deprivation in anesthetized rats was similar in timing to that of controls. No delayed rebound effects were observed in either group for 72 h after deprivation. These data show that a recovery process similar to that occurring during naturally occurring sleep also takes place during anesthesia and suggest that sleep and anesthesia share common regulatory mechanisms. Such interactions between sleep and anesthesia may allow anesthesiologists to better understand a potentially important source of variability in anesthetic action and raise the possibility that anesthetics may facilitate sleep in environments where sleep deprivation is common.
Article
General anaesthetics cause sedation, amnesia and hypnosis. Although these clinically desired actions are indicative of an impairment of neocortical information processing, it is widely held that they are to a large part mediated by subcortical neural networks. Anaesthetic action on brain stem, basal forebrain and thalamus, all of which are known to modulate cortical excitability, would thus ultimately converge on neocortex, perturbing and reducing action potential activity therein. However, as neocortex harbours molecular targets of anaesthetics in high densities, notably GABA(A) receptors, neocortex itself should be very sensitive to anaesthetics. Here, we performed experiments to reveal the extent to which neocortex proper is a relevant target of the low concentrations of volatile anaesthetics causing sedation and hypnosis. We compared the effects of isoflurane, enflurane and halothane on spontaneous action potential activity of rat neocortical neurons in vivo and in isolated cortical networks in vitro, i.e. in the presence and absence of subcortical arousal systems. We observed that the anaesthetics decreased spontaneous firing of neurons via intracortical mechanisms; concentrations inducing hypnosis in humans reduced discharge rates both in vivo and in vitro to the same extent, approximately 50%. This decrease in neuronal activity was paralleled by a significant enhancement of neocortical GABA(A) receptor-mediated inhibition. These findings challenge the notion of predominantly subcortical effects of volatile anaesthetics and suggest that intracortical targets, among them neocortical GABA(A) receptors, mediate the sedative and hypnotic properties of volatile anaesthetics.
Article
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.
Article
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.
Response properties of neurons of cat’s somatic sensory cortex to peripheral stimuli
  • Mountcastle
The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway
  • Nelson