The Modulation by Anesthetics and Analgesics of Respiratory Rhythm in the Nervous System.

Rhythmic eupneic breathing in mammals depends on the coordinated activities of the neural system that sends cranial and spinal motor outputs to respiratory muscles. These outputs modulate lung ventilation and adjust respiratory airflow, which depends on the upper airway patency and ventilatory musculature. Anesthetics are widely used in clinical practice worldwide. In addition to clinically necessary pharmacological effects, respiratory depression is a critical side effect induced by most general anesthetics. Therefore, understanding how general anesthetics modulate the respiratory system is important for the development of safer general anesthetics. Currently used volatile anesthetics and most intravenous anesthetics induce inhibitory effects on respiratory outputs. Various general anesthetics produce differential effects on respiratory characteristics, including the respiratory rate, tidal volume, airway resistance, and ventilatory response. At the cellular and molecular levels, the mechanisms underlying anesthetic-induced breathing depression mainly include modulation of synaptic transmission of ligand-gated ionotropic receptors (e.g., γ-aminobutyric acid, N-methyl-D-aspartate, and nicotinic acetylcholine receptors) and ion channels (e.g., voltage-gated sodium, calcium, and potassium channels, two-pore domain potassium channels, and sodium leak channels), which affect neuronal firing in brainstem respiratory and peripheral chemoreceptor areas. The present review comprehensively summarizes the modulation of the respiratory system by clinically used general anesthetics, including the effects at the molecular, cellular, anatomic, and behavioral levels. Specifically, analgesics, such as opioids, which cause respiratory depression and the "opioid crisis", are discussed. Finally, underlying strategies of respiratory stimulation that target general anesthetics and/or analgesics are summarized.

Crosstalk between GABAA receptors in astrocytes and neurons triggered by general anesthetic drugs.

General anesthetic drugs cause cognitive deficits that persist after the drugs have been eliminated. Astrocytes may contribute to such cognition-impairing effects through the release of one or more paracrine factors that increase a tonic inhibitory conductance generated by extrasynaptic γ-aminobutyric acid type A (GABAA) receptors in hippocampal neurons. The mechanisms underlying this astrocyte-to-neuron crosstalk remain unknown. Interestingly, astrocytes express anesthetic-sensitive GABAA receptors. Here, we tested the hypothesis that anesthetic drugs activate astrocytic GABAA receptors to initiate crosstalk leading to a persistent increase in extrasynaptic GABAA receptor function in neurons. We also investigated the signaling pathways in neurons and aimed to identify the paracrine factors released from astrocytes. Astrocytes and neurons from mice were grown in primary cell cultures and studied using in vitro electrophysiological and biochemical assays. We discovered that the commonly used anesthetics etomidate (injectable) and sevoflurane (inhaled) stimulated astrocytic GABAA receptors, which in turn promoted the release paracrine factors, that increased the tonic current in neurons via a p38 MAPK-dependent signaling pathway. The increase in tonic current was mimicked by exogenous IL-1ß and abolished by blocking IL-1 receptors; however, unexpectedly, IL-1ß and other cytokines were not detected in astrocyte-conditioned media. In summary, we have identified a novel form of crosstalk between GABAA receptors in astrocytes and neurons that engages a p38 MAPK-dependent pathway. Brief commentary BACKGROUND: Many older patients experience cognitive deficits after surgery. Anesthetic drugs may be a contributing factor as they cause a sustained increase in the function of "memory blocking" extrasynaptic GABAA receptors in neurons. Interestingly, astrocytes are required for this increase; however, the mechanisms underlying the astrocyte-to-neuron crosstalk remain unknown. TRANSLATIONAL SIGNIFICANCE: We discovered that commonly used general anesthetic drugs stimulate GABAA receptors in astrocytes, which in turn release paracrine factors that trigger a persistent increase in extrasynaptic GABAA receptor function in neurons via p38 MAPK. This novel form of crosstalk may contribute to persistent cognitive deficits after general anesthesia and surgery.

Anesthetic Binding Induced Motion of GABAA Receptors Revealed by Coarse-Grained Molecular Dynamics Simulations.

General anesthetics are indispensable in modern medicine because they induce a reversible loss of consciousness and sensation in humans. On the other hand, their molecular mechanisms of action have not yet been elucidated. Several studies have identified the main targets of some general anesthetics. The structures of γ-aminobutyric acid A (GABAA) receptors with the intravenous anesthetics such as propofol and etomidate have recently been determined. Although these anesthetic binding structures provide essential insights into the mechanism of action of anesthetics, the detailed molecular mechanism of how the anesthetic binding affects the Cl- permeability of GABAA receptors remains to be elucidated. In this study, we performed coarse-grained molecular dynamics simulations for GABAA receptors and analyzed the resulting simulation trajectories to investigate the effects of anesthetic binding on the motion of GABAA receptors. The results showed large structural fluctuations in GABAA receptors, correlations of motion between the amino acid residues, large amplitude motion, and autocorrelated slow motion, which were obtained by advanced statistical analyses. In addition, a comparison of the resulting trajectories in the presence or absence of the anesthetic molecules revealed a characteristic pore motion related to the gate-opening motion of GABAA receptors.

Isoflurane Rapidly Modifies Synaptic and Cytoskeletal Phosphoproteomes of the Supraoptic Nucleus of the Hypothalamus and the Cortex.

INTRODUCTION: Despite the widespread use of general anaesthetics, the mechanisms mediating their effects are still not understood. Although suppressed in most parts of the brain, neuronal activity, as measured by FOS activation, is increased in the hypothalamic supraoptic nucleus (SON) by numerous general anaesthetics, and evidence points to this brain region being involved in the induction of general anaesthesia (GA) and natural sleep. Posttranslational modifications of proteins, including changes in phosphorylation, enable fast modulation of protein function which could be underlying the rapid effects of GA. In order to identify potential phosphorylation events in the brain-mediating GA effects, we have explored the phosphoproteome responses in the rat SON and compared these to cingulate cortex (CC) which displays no FOS activation in response to general anaesthetics. METHODS: Adult Sprague-Dawley rats were treated with isoflurane for 15 min. Proteins from the CC and SON were extracted and processed for nano-LC mass spectrometry (LC-MS/MS). Phosphoproteomic determinations were performed by LC-MS/MS. RESULTS: We found many changes in the phosphoproteomes of both the CC and SON in response to 15 min of isoflurane exposure. Pathway analysis indicated that proteins undergoing phosphorylation adaptations are involved in cytoskeleton remodelling and synaptic signalling events. Importantly, changes in protein phosphorylation appeared to be brain region specific suggesting that differential phosphorylation adaptations might underlie the different neuronal activity responses to GA between the CC and SON. CONCLUSION: In summary, these data suggest that rapid posttranslational modifications in proteins involved in cytoskeleton remodelling and synaptic signalling events might mediate the central mechanisms mediating GA.

Structural interplay of anesthetics and paralytics on muscle nicotinic receptors.

General anesthetics and neuromuscular blockers are used together during surgery to stabilize patients in an unconscious state. Anesthetics act mainly by potentiating inhibitory ion channels and inhibiting excitatory ion channels, with the net effect of dampening nervous system excitability. Neuromuscular blockers act by antagonizing nicotinic acetylcholine receptors at the motor endplate; these excitatory ligand-gated ion channels are also inhibited by general anesthetics. The mechanisms by which anesthetics and neuromuscular blockers inhibit nicotinic receptors are poorly understood but underlie safe and effective surgeries. Here we took a direct structural approach to define how a commonly used anesthetic and two neuromuscular blockers act on a muscle-type nicotinic receptor. We discover that the intravenous anesthetic etomidate binds at an intrasubunit site in the transmembrane domain and stabilizes a non-conducting, desensitized-like state of the channel. The depolarizing neuromuscular blocker succinylcholine also stabilizes a desensitized channel but does so through binding to the classical neurotransmitter site. Rocuronium binds in this same neurotransmitter site but locks the receptor in a resting, non-conducting state. Together, this study reveals a structural mechanism for how general anesthetics work on excitatory nicotinic receptors and further rationalizes clinical observations in how general anesthetics and neuromuscular blockers interact.

Understanding the Molecular Mechanism of Anesthesia: Effect of General Anesthetics and Structurally Similar Non-Anesthetics on the Properties of Lipid Membranes.

General anesthesia can be caused by various, chemically very different molecules, while several other molecules, many of which are structurally rather similar to them, do not exhibit anesthetic effects at all. To understand the origin of this difference and shed some light on the molecular mechanism of general anesthesia, we report here molecular dynamics simulations of the neat dipalmitoylphosphatidylcholine (DPPC) membrane as well as DPPC membranes containing the anesthetics diethyl ether and chloroform and the structurally similar non-anesthetics n-pentane and carbon tetrachloride, respectively. To also account for the pressure reversal of anesthesia, these simulations are performed both at 1 bar and at 600 bar. Our results indicate that all solutes considered prefer to stay both in the middle of the membrane and close to the boundary of the hydrocarbon domain, at the vicinity of the crowded region of the polar headgroups. However, this latter preference is considerably stronger for the (weakly polar) anesthetics than for the (apolar) non-anesthetics. Anesthetics staying in this outer preferred position increase the lateral separation between the lipid molecules, giving rise to a decrease of the lateral density. The lower lateral density leads to an increased mobility of the DPPC molecules, a decreased order of their tails, an increase of the free volume around this outer preferred position, and a decrease of the lateral pressure at the hydrocarbon side of the apolar/polar interface, a change that might well be in a causal relation with the occurrence of the anesthetic effect. All these changes are clearly reverted by the increase of pressure. Furthermore, non-anesthetics occur in this outer preferred position in a considerably smaller concentration and hence either induce such changes in a much weaker form or do not induce them at all.

Inhibition of mitochondrial respiration by general anesthetic drugs.

General anesthetic drugs have been associated with various unwanted effects including an interference with mitochondrial function. We had previously observed increases of lactate formation in the mouse brain during anesthesia with volatile anesthetic agents. In the present work, we used mitochondria that were freshly isolated from mouse brain to test mitochondrial respiration and ATP synthesis in the presence of six common anesthetic drugs. The volatile anesthetics isoflurane, halothane, and (to a lesser extent) sevoflurane caused an inhibition of complex I of the electron transport chain in a dose-dependent manner. Significant effects were seen at concentrations that are reached under clinical conditions (< 0.5 mM). Pentobarbital and propofol also inhibited complex I but at concentrations that were two-fold higher than clinical EC50 values. Only propofol caused an inhibition of complex II. Complex IV respiration was not affected by either agent. Ketamine did not affect mitochondrial respiration. Similarly, all anesthetic agents except ketamine suppressed ATP production at high concentrations. Only halothane increased cytochrome c release indicating damage of the mitochondrial membrane. In summary, volatile general anesthetic agents as well as pentobarbital and propofol dose-dependently inhibit mitochondrial respiration. This action may contribute to depressive actions of the drugs in the brain.

Claustrum modulates behavioral sensitivity and EEG activity of propofol anesthesia.

AIMS: The claustrum has long been regarded as a vital center for conscious control. Electrical stimulation or damage to the claustrum can result in decreased awareness or loss of consciousness, suggesting that the claustrum may be a target for the action of general anesthetics. This study aimed to determine the role of the claustrum in propofol anesthesia. METHODS: We first applied a fiber photometry calcium signal recording system to record the claustral neuronal activity during the entire process of propofol anesthesia. Chemogenetic activation of claustral neurones was then performed to verify their role in anesthesia. Finally, muscimol (GABAa receptor agonist) and gabazine (GABAa receptor antagonist) were microinjected into the claustrum to determine whether their GABAa receptors were involved in modulating propofol anesthesia. EEG and behavioral indicators, such as anesthetic sensitivity and efficacy, were recorded and analyzed. RESULTS: An evident anesthesia-related change in claustrum neuronal activity was suppressed during propofol-induced unconsciousness and restored following recovery from anesthesia. Chemogenetic activation of claustrum neurons results in attenuated propofol sensitivity, a shorter anesthesia duration, and an EEG shift toward wakefulness. Manipulation of GABAa receptors in the claustrum showed bidirectional control of propofol sensitivity, as activation decreases anesthesia efficiency while inactivation augments it. Additionally, inhibiting claustrum GABAa receptors increases cortical EEG slow waves. CONCLUSIONS: Claustrum neurones and their GABAa receptors are implicated in the modulation of propofol anesthesia in both behavioral and EEG assessments. Our findings create scope to reveal the brain targets of anesthetic action further and add to the existing evidence on the consciousness-modulating role of the claustrum.

Hypoxia-triggered O-GlcNAcylation in the brain drives the glutamate-glutamine cycle and reduces sensitivity to sevoflurane in mice.

BACKGROUND: Hypersensitivity to general anaesthetics predicts adverse postoperative outcomes in patients. Hypoxia exerts extensive pathophysiological effects on the brain; however, whether hypoxia influences sevoflurane sensitivity and its underlying mechanisms remain poorly understood. METHODS: Mice were acclimated to hypoxia (oxygen 10% for 8 h day-1) for 28 days and anaesthetised with sevoflurane; the effective concentrations for 50% of the animals (EC50) showing loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR) were determined. Positron emission tomography-computed tomography, O-glycoproteomics, seahorse analysis, carbon-13 tracing, site-specific mutagenesis, and electrophysiological techniques were performed to explore the underlying mechanisms. RESULTS: Compared with the control group, the hypoxia-acclimated mice required higher concentrations of sevoflurane to present LORR and LTWR (EC50LORR: 1.61 [0.03]% vs 1.46 [0.04]%, P<0.01; EC50LTWR: 2.46 [0.14]% vs 2.22 [0.06]%, P<0.01). Hypoxia-induced reduction in sevoflurane sensitivity was correlated with elevation of protein O-linked N-acetylglucosamine (O-GlcNAc) modification in brain, especially in the thalamus, and could be abolished by 6-diazo-5-oxo-l-norleucine, a glutamine fructose-6-phosphate amidotransferase inhibitor, and mimicked by thiamet-G, a selective O-GlcNAcase inhibitor. Mechanistically, O-GlcNAcylation drives de novo synthesis of glutamine from glucose in astrocytes and promotes the glutamate-glutamine cycle, partially via glycolytic flux and activation of glutamine synthetase. CONCLUSIONS: Intermittent hypoxia exposure decreased mouse sensitivity to sevoflurane anaesthesia through enhanced O-GlcNAc-dependent modulation of the glutamate-glutamine cycle in the brain.

The general anaesthetic propofol prevents cerebrocortical potentiation in neocortical mouse brain slices.

Propofol is well known to cause amnesia independent of its sedative effect. Memory consolidation processes in the hippocampus have been proposed as a target - however the neural substrates for propofol's amnesic actions remain understudied and poorly described. In particular, the potential role of the cerebral cortex has not been investigated. As an in vitro experimental model of cortical memory consolidation, potentiated cerebral cortex evoked responses were generated in mouse neocortical slices using high frequency (20 Hz) stimulation to layer IV cortical grey matter or subcortical white matter. In separate experiments, slices were pretreated with propofol at two concentrations, 2 µg/mL and 4 µg/mL, to determine the effect of clinically relevant propofol levels on the potentiation response. Only grey matter stimulation induced a significant and lasting increase in cortical evoked potential amplitude in the drug-free condition. Propofol at 2 µg/mL completely inhibited cortical evoked response potentiation, while the 4 µg/mL concentration caused a small but significant depressant effect consequent to the high frequency stimulation. These findings support the hypothesis that propofol disrupts memory consolidation and actively facilitates memory decay in the cerebral cortex. The results further highlight the importance of the cerebral cortex in the early phase of long term memory consolidation.

Modeling cortical synaptic effects of anesthesia and their cholinergic reversal.

General anesthetics work through a variety of molecular mechanisms while resulting in the common end point of sedation and loss of consciousness. Generally, the administration of common anesthetics induces reduction in synaptic excitation while promoting synaptic inhibition. Exogenous modulation of the anesthetics' synaptic effects can help determine the neuronal pathways involved in anesthesia. For example, both animal and human studies have shown that exogenously induced increases in acetylcholine in the brain can elicit wakeful-like behavior despite the continued presence of the anesthetic. However, the underlying mechanisms of anesthesia reversal at the cellular level have not been investigated. Here we apply a computational model of a network of excitatory and inhibitory neurons to simulate the network-wide effects of anesthesia, due to changes in synaptic inhibition and excitation, and their reversal by cholinergic activation through muscarinic receptors. We use a differential evolution algorithm to fit model parameters to match measures of spiking activity, neuronal connectivity, and network dynamics recorded in the visual cortex of rodents during anesthesia with desflurane in vivo. We find that facilitating muscarinic receptor effects of acetylcholine on top of anesthetic-induced synaptic changes predicts the reversal of anesthetic suppression of neurons' spiking activity, functional connectivity, as well as pairwise and population interactions. Thus, our model predicts a specific neuronal mechanism for the cholinergic reversal of anesthesia consistent with experimental behavioral observations.

General anesthesia and the cortical stranglehold on consciousness.

In this issue of Neuron, Bharioke et al. (2022) demonstrate that diverse general anesthetic regimens all reversibly and selectively synchronize spontaneous activity of pyramidal neurons in layer 5 of mouse cortex. We discuss the implications of these findings for the mechanism of consciousness and anesthetic-induced unconsciousness.

General anesthesia globally synchronizes activity selectively in layer 5 cortical pyramidal neurons.

General anesthetics induce loss of consciousness, a global change in behavior. However, a corresponding global change in activity in the context of defined cortical cell types has not been identified. Here, we show that spontaneous activity of mouse layer 5 pyramidal neurons, but of no other cortical cell type, becomes consistently synchronized in vivo by different general anesthetics. This heightened neuronal synchrony is aperiodic, present across large distances, and absent in cortical neurons presynaptic to layer 5 pyramidal neurons. During the transition to and from anesthesia, changes in synchrony in layer 5 coincide with the loss and recovery of consciousness. Activity within both apical and basal dendrites is synchronous, but only basal dendrites' activity is temporally locked to somatic activity. Given that layer 5 is a major cortical output, our results suggest that brain-wide synchrony in layer 5 pyramidal neurons may contribute to the loss of consciousness during general anesthesia.

Thalamic T-Type Calcium Channels as Targets for Hypnotics and General Anesthetics.

General anesthetics mainly act by modulating synaptic inhibition on the one hand (the potentiation of GABA transmission) or synaptic excitation on the other (the inhibition of NMDA receptors), but they can also have effects on numerous other proteins, receptors, and channels. The effects of general anesthetics on ion channels have been the subject of research since the publication of reports of direct actions of these drugs on ion channel proteins. In particular, there is considerable interest in T-type voltage-gated calcium channels that are abundantly expressed in the thalamus, where they control patterns of cellular excitability and thalamocortical oscillations during awake and sleep states. Here, we summarized and discussed our recent studies focused on the CaV3.1 isoform of T-channels in the nonspecific thalamus (intralaminar and midline nuclei), which acts as a key hub through which natural sleep and general anesthesia are initiated. We used mouse genetics and in vivo and ex vivo electrophysiology to study the role of thalamic T-channels in hypnosis induced by a standard general anesthetic, isoflurane, as well as novel neuroactive steroids. From the results of this study, we conclude that CaV3.1 channels contribute to thalamocortical oscillations during anesthetic-induced hypnosis, particularly the slow-frequency range of δ oscillations (0.5-4 Hz), by generating "window current" that contributes to the resting membrane potential. We posit that the role of the thalamic CaV3.1 isoform of T-channels in the effects of various classes of general anesthetics warrants consideration.

Effects of General Anesthetics on Synaptic Transmission and Plasticity.

General anesthetics depress excitatory and/or enhance inhibitory synaptic transmission principally by modulating the function of glutamatergic or GABAergic synapses, respectively, with relative anesthetic agent-specific mechanisms. Synaptic signaling proteins, including ligand- and voltage-gated ion channels, are targeted by general anesthetics to modulate various synaptic mechanisms, including presynaptic neurotransmitter release, postsynaptic receptor signaling, and dendritic spine dynamics to produce their characteristic acute neurophysiological effects. As synaptic structure and plasticity mediate higher-order functions such as learning and memory, long-term synaptic dysfunction following anesthesia may lead to undesirable neurocognitive consequences depending on the specific anesthetic agent and the vulnerability of the population. Here we review the cellular and molecular mechanisms of transient and persistent general anesthetic alterations of synaptic transmission and plasticity.

Identifying c-fos Expression as a Strategy to Investigate the Actions of General Anesthetics on the Central Nervous System.

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 a c-fos expression.

General anaesthetics reduce acute lymphoblastic leukaemia malignancies in vitro and in vivovia CXCR4 and osteopontin mediated mechanisms.

Background: Acute lymphoblastic leukaemia (ALL) is a common type of cancer in children. General anaesthetics are often used on patients undergoing painful procedures during ALL treatments but their effects on ALL malignancy remain unknown. Herein, we aim to study the effect of propofol and sevoflurane on the migration, homing and chemoresistance of ALL cells. Methods: NALM-6 and Reh cells were treated with propofol (5 and 10 µg/ml) or sevoflurane (3.6%) in vitro for six hours. Then, cells were harvested for adhesion assay and migration assay in vitro. In in vivo experiments, GFP-NALM-6 cells were pre-treated with propofol (10 µg/ml) or sevoflurane (3.6%) for six hours. Then, cells were injected intravenously to C57BL/6 female mice followed by intravital microscopy. For chemoresistance study, cells were treated with rising concentrations of Ara-c (0.05-50 nM) plus 10µg/ml of propofol or Ara-C plus 3.6% of sevoflurane for 4 hours, followed by the assessment of cell viability via CCK-8 assay and detection of autophagy via flow cytometry. Results: Both anaesthetics reduced in vivo migration and in vivo homing as exemplified by 1) the reduction in the number of cells entering the bone marrow and 2) the disturbance in homing location in relation to endosteal surface. Our results indicated that general anaesthetics reduced the surface CXCR4 expression and the adhesion of leukaemia cells to thrombin cleaved osteopontin (OPN) was reduced. Those changes might result in the alterations in migration and homing. In addition, both anaesthetics sensitised ALL cells to Ara-c possibly through CXCR4 mediated mechanisms. Propofol but not sevoflurane enhanced chemo-related cell death via inducing cytotoxic autophagy. Conclusion: Together, our data suggest that both propofol and sevoflurane could reduce ALL migration, and homing in vivo and in vitro via CXCR4 and OPN mediated mechanisms. Both anaesthetics could sensitise ALL cells to chemotherapy possibly via CXCR4 mediated mechanisms.

Ketamine-xylazine anesthesia depth affects auditory neuronal responses in the lateral superior olive complex of the gerbil.

Studies of in vivo neuronal responses to auditory inputs in the superior olive complex (SOC) are usually done under anesthesia. However, little attention has been paid to the effect of anesthesia itself on response properties. Here, we assessed the effect of anesthesia depth under ketamine-xylazine anesthetics on auditory evoked response properties of lateral SOC neurons. Anesthesia depth was tracked by monitoring EEG spectral peak frequencies. An increase in anesthesia depth led to a decrease of spontaneous discharge activities and an elevated response threshold. The temporal responses to suprathreshold tones were also affected, with adapted responses reduced but peak responses unaffected. Deepening the anesthesia depth also increased first spike latency. However, spike jitter was not affected. Auditory brainstem responses to clicks confirmed that ketamine-xylazine anesthesia depth affects auditory neuronal activities and the effect on spike rate and spike timing persists through the auditory pathway. We concluded from those observations that ketamine-xylazine affects lateral SOC response properties depending on the anesthesia depth.NEW & NOTEWORTHY We studied how the depth of ketamine-xylazine anesthesia altered response properties of lateral superior olive complex neurons, and auditory brainstem evoked responses. Our results provide direct evidence that anesthesia depth affects auditory neuronal responses and reinforce the notion that both the anesthetics and the anesthesia depth should be considered when interpreting/comparing in vivo neuronal recordings.