Genetic effects on an anesthetic-sensitive pathway in the brain of Drosophila

General anesthetics are known to inhibit the electrically induced escape response of the fruitfly through action within the brain. We examined this response and its sensitivity to anesthetics in several mutants that cause significant disruption of the mushroom body and other structures of the central brain in adult flies. Because we show here that anesthesia sensitivity is influenced by genetic background, we have used a set of congenic mutant lines. Sensitivity to halothane is normal in most of these lines, indicating that the anesthetic target is unaffected by the gross status of the central brain. Thus, for the escape response, anesthetic sensitivity is not a global feature but reflects action at a localized target. Only the mushroom body defect (mud) line showed an increased sensitivity of the escape response to halothane. Sensitivity to two other anesthetics is also perturbed in this line, albeit less dramatically so. The behavior of mud/+ heterozygotes and the comparison of brain anatomy among all the mutant lines imply that the effect of the mud mutation on anesthesia is not via gross alteration of central brain structures. The possibility that an adventitious mutation in the mud line is responsible for the effects on anesthesia is disfavored by the behavior of a heterozygote between two mud alleles. Although we do not yet know whether the mud gene encodes an anesthetic target or influences the functioning of an anesthetic-sensitive neuron in this pathway, our work indicates that this gene regulates the effects of halothane on a circumscribed pathway.     

A Genetic and Mosaic Analysis of a Locus Involved in the Anesthesia Response of Drosophila Melanogaster

We describe a genetic and behavioral analysis of several alleles of har38, a mutant with altered sensitivity to the general anesthetic halothane. We obtained a P-element-induced allele of har38 and generated several excision alleles by remobilizing the P element. The mutants narrow abdomen (na) and har85 are confirmed to be allelic to har38. Besides a decreased sensitivity to halothane, all mutant alleles of this locus cause a characteristic walking behavior in the absence of anesthetics. We have quantified this behavior using a geotaxis apparatus. Responses of the mutant alleles to different inhalational anesthetics were tested. The results strongly favor a multipathway model for the onset of anesthesia. Mosaic flies were tested for their response to halothane and checked for their abnormal walking behavior. The analysis suggests that both the behaviors are exhibited only by such mosaics as have the entire head of mutant origin. It is likely that this focus represents an element of a common pathway in the anesthetic response to several inhalational anesthetics but not all. This result is the first demonstration of regional specificity in the CNS of any animal for general anesthetic action.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The ¹⁹F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the ¹⁹F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, ¹⁹F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo ¹⁹F-NMR surface coil and imaging studies.     

The effect in vitro of volatile anesthetics on the activity of cholinesterases.

Because the mechanism of anesthesia is unknown, the relationship between anesthetics and enzymes essential to brain function may be an important one. Therefore, the effect of 8 volatile anesthetics on the enzymatic activity of solubilized, purified dog brain and human erythrocyte acetylcholinesterase (AChE) and human serum cholinesterase (ChE) was studied in vitro. Serum ChE was found to be insensitive to saturated solutions of all the anesthetics studied. However, brain and erythrocyte AChE were reversibly inhibited in a dose-dependent manner by all 8 anesthetics in concentrations exceeding those used in clinical practice. Kinetic analysis revealed a mixed (competitive, non-competitive) type of inhibition with the exception of the ether-crythrocyte AChE interaction which was characterized by competitive inhibition. Ether and methoxyflurane were found to depress the AChE activity the most and isoflurane and enflurane the least. The concentrations of anesthetic in the gas phase necessary for 50% inhibition of erythrocyte AChE activity (I₅₀) were calculated for 5 anesthetics and found to correlate with their water-gas partition coefficients. These data suggest that the effect in vitro of volatile anesthetics on the catalytic activity of cholinesterases is a variable one and may be unrelated to anesthetic potency in vivo. The implications of these data concerning anesthetic-active site interactions are discussed.     

L-phenylisopropyladenosine (L-PIA) diminishes halothane anesthetic requirements and decreases noradrenergic neurotransmission in rats

The effect of L-phenylisopropyladenosine (L-PIA), the A1 adenosine agonist, on the depth of anesthesia was investigated in halothane-anesthetized rats. L-PIA treatment reduced the minimum anesthetic concentration (MAC) of halothane that prevented 50% of animals from moving in response to a painful stimulus by 49%. MAC experiments performed with L-PIA given in conjunction with A1 adenosine receptor antagonists which either permeate the blood-brain barrier (8-phenyltheophylline [8-PT] or do not (8-sulphophenyltheophylline [8-So-PT]) indicate that central mechanisms are involved. Noradrenergic neurotransmission was diminished following L-PIA administration in halothane-anesthetized rats in all brain regions. These data suggest that acute L-PIA treatment decreases central noradrenergic neurotransmission and may represent the mechanism for the decrease in halothane dose to achieve an anesthetic endpoint anesthetic response to halothane.     

Molecular dynamics simulations of C2F6 effects on gramicidin A: implications of the mechanisms of general anesthesia

It was recently postulated that the effects of general anesthetics on protein global dynamics might underlie a unitary molecular mechanism of general anesthesia. To verify that the specific dynamics effects caused by general anesthetics were not shared by nonanesthetic molecules, two parallel 8-ns all-atom molecular dynamics simulations were performed on a gramicidin A (gA) channel in a fully hydrated dimyristoylphosphatidylcholine membrane in the presence and absence of hexafluoroethane (HFE), which structurally resembles the potent anesthetic molecule halothane but produces no anesthesia. Similar to halothane, HFE had no measurable effects on the gA channel structure. In contrast to halothane, HFE produced no significant changes in the gA channel dynamics. The difference between halothane and HFE on channel dynamics can be attributed to their distinctly different distributions within the lipid bilayer and consequently to the different interactions of the anesthetic and the nonanesthetic molecules with the channel-anchoring tryptophan residues. The study further supports the notion that anesthetic-induced changes in protein global dynamics may play an important role in mediating anesthetic actions on proteins.     

The effects of halothane and isoflurane on intracellular Ca2+ regulation in cultured cells with characteristics of vascular smooth muscle.

The Ca(2+)-sensitive photoprotein aequorin was used to monitor changes in intracellular [Ca2+] within cultured cells with characteristics of vascular smooth muscle. Two cell lines were investigated: they were A10 cells, which are transformed cells originally derived from rat aorta, and BC3H1 cells obtained from mouse brain neoplasm. Transient increases in intracellular [Ca2+] were induced following exposure to two different volatile anaesthetics (halothane and isoflurane) and various vasoactive substances (acetylcholine, endothelin, histamine, serotonin and vasopressin). The amplitude of the transients induced by isoflurane were more dependent on the presence of extracellular Ca2+ than those induced by halothane, thus the modes and/or locations of action of these two anesthetics are somewhat different. The response of the two cell lines to the vasoactive substances are unique. Receptor activated changes in [Ca2+]i by various agonists were diminished in the presence and absence of either anesthetic. These data suggest that, although the receptor populations within each cell line were slightly different, the prior application of a volatile anesthetic in a clinically-relevant dose induced a transient increase in [Ca2+]i that could subsequently diminish agonist responses.     

Optical reversal of halothane-induced immobility in C. elegans

Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [ [1] and [2] ]. Genetic studies in Caenorhabditis elegans [ [3] and [4] ], Drosophila [ [3] and [5] ], and mice [ [6] , [7] , [8] and [9] ] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K⁺ [ [10] , [11] , [12] and [13] ] or permeable to Na⁺ [14] are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane-induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of nonspecific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.     

Altered anesthetic sensitivity of mice lacking ndufs4, a subunit of mitochondrial complex I

Anesthetics are in routine use, yet the mechanisms underlying their function are incompletely understood. Studies in vitro demonstrate that both GABA(A) and NMDA receptors are modulated by anesthetics, but whole animal models have not supported the role of these receptors as sole effectors of general anesthesia. Findings in C. elegans and in children reveal that defects in mitochondrial complex I can cause hypersensitivity to volatile anesthetics. Here, we tested a knockout (KO) mouse with reduced complex I function due to inactivation of the Ndufs4 gene, which encodes one of the subunits of complex I. We tested these KO mice with two volatile and two non-volatile anesthetics. KO and wild-type (WT) mice were anesthetized with isoflurane, halothane, propofol or ketamine at post-natal (PN) days 23 to 27, and tested for loss of response to tail clamp (isoflurane and halothane) or loss of righting reflex (propofol and ketamine). KO mice were 2.5 - to 3-fold more sensitive to isoflurane and halothane than WT mice. KO mice were 2-fold more sensitive to propofol but resistant to ketamine. These changes in anesthetic sensitivity are the largest recorded in a mammal.     

Lactate levels in the brain are elevated upon exposure to volatile anesthetics: a microdialysis study

Anesthetic agents have well-defined pharmacological targets but their effects on energy metabolism in the brain are poorly understood. In this study, we examined the effects of different anesthetics on extracellular lactate and glucose levels in blood, CSF and brain of the mouse. In vivo-microdialysis was used to monitor extracellular energy metabolites in the brain of awake mice and during anesthesia with seven different anesthetic drugs. In separate groups, lactate and glucose concentrations in blood and CSF were measured for each anesthetic. We found that anesthesia with isoflurane caused a large increase of extracellular lactate levels in mouse striatum and hippocampus (300-400%). Pyruvate levels also increased while glucose and glutamate levels were unchanged. This effect was dose-dependent and was mimicked by other gaseous anesthetics such as halothane and sevoflurane but not by intravenous anesthetics. Ketamine/xylazine and chloral hydrate caused 2-fold increases of glucose levels in mouse blood and brain while lactate levels were only moderately increased. Propofol caused a minor increase of extracellular glucose levels while pentobarbital had no effect on either lactate or glucose. Volatile anesthetics also increased lactate levels in blood and CSF by 2-3-fold but had no effect on plasma glucose. Further experiments demonstrated that lactate formation by isoflurane in mouse brain was independent of neuronal impulse flow and did not involve ATP-dependent potassium channels. We conclude that volatile anesthetics, but not intravenous anesthetics, cause a specific, dose-dependent increase in extracellular lactate levels in mouse brain. This effect occurs in the absence of ischemia, is independent of peripheral actions and is reflected in strongly increased CSF lactate levels.     

Lactate levels in the brain are elevated upon exposure to volatile anesthetics: A microdialysis study

Anesthetic agents have well-defined pharmacological targets but their effects on energy metabolism in the brain are poorly understood. In this study, we examined the effects of different anesthetics on extracellular lactate and glucose levels in blood, CSF and brain of the mouse. In vivo-microdialysis was used to monitor extracellular energy metabolites in the brain of awake mice and during anesthesia with seven different anesthetic drugs. In separate groups, lactate and glucose concentrations in blood and CSF were measured for each anesthetic. We found that anesthesia with isoflurane caused a large increase of extracellular lactate levels in mouse striatum and hippocampus (300–400%). Pyruvate levels also increased while glucose and glutamate levels were unchanged. This effect was dose-dependent and was mimicked by other gaseous anesthetics such as halothane and sevoflurane but not by intravenous anesthetics. Ketamine/xylazine and chloral hydrate caused 2-fold increases of glucose levels in mouse blood and brain while lactate levels were only moderately increased. Propofol caused a minor increase of extracellular glucose levels while pentobarbital had no effect on either lactate or glucose. Volatile anesthetics also increased lactate levels in blood and CSF by 2–3-fold but had no effect on plasma glucose. Further experiments demonstrated that lactate formation by isoflurane in mouse brain was independent of neuronal impulse flow and did not involve ATP-dependent potassium channels. We conclude that volatile anesthetics, but not intravenous anesthetics, cause a specific, dose-dependent increase in extracellular lactate levels in mouse brain. This effect occurs in the absence of ischemia, is independent of peripheral actions and is reflected in strongly increased CSF lactate levels.     

Die neuronale Grundlage des Zustandes der Narkose: ein vergleichend-physiologischer Ansatz

The sensitivity of flies and locusts to halothane and N₂O was investigated. In this paper we report experiments concerning the allover motor activity in the animal as a whole. In order to determine how the size of neurons comes into play under anesthesia we experimented with different but closely related species of flies differing very clearly in size. For the same reason we chose locusts of different developmental states and consequently different size. It came out that the larger insects are more sensitive to anesthetics than the smaller ones. The results confirm one of Sherrington's (1906) conclusions, which says the axon which conducts spikes cannot be the most sensitive part of the neuron to anesthetic action. He ascribed the highest sensitivity to synapses; this, however, does not match with our results. In agreement with our experimental data is the new hypothesis that long dendrites or axonal endings conducting graded potentials are those parts of the CNS that exhibit the highest sensitivity to anesthetic action. Further confirmation of this hypothesis by more direct approaches has to be provided.     

Lack of enantiomeric specificity in the effects of anesthetic steroids on lipid bilayers

The most important target protein for many anesthetics, including volatile and steroid anesthetics, appears to be the type A γ-amino butyric acid receptor (GABA_AR), yet direct binding remains to be demonstrated. Hypotheses of lipid-mediated anesthesia suggest that lipid bilayer properties are changed by anesthetics and that this in turn affects the functions of proteins. While other data could equally well support direct or lipid-mediated action, enantiomeric specificity displayed by some anesthetics is not reflected in their interactions with lipids. In the present study, we studied the effects of two pairs of anesthetic steroid enantiomers on bilayers of several compositions, measuring potentially relevant physical properties. For one of the pairs, allopregnanolone and ent-allopregnanolone, the natural enantiomer is 300% more efficacious as an anesthetic, while for the other, pregnanolone and ent-pregnanolone, there is little difference in anesthetic potency. For each enantiomer pair, we could find no differences. This strongly favors the view that the effects of these anesthetics on lipid bilayers are not relevant for the main features of anesthesia. These steroids also provide tools to distinguish in general the direct binding of steroids to proteins from lipid-mediated effects.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The 19F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the 19F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, 19F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo 19F-NMR surface coil and imaging studies.     

Antagonism between high pressure and anesthetics in the thermal phase-transition of dipalmitoyl phosphatidylcholine bilayer

The antagonizing action of hydrostatic pressure against anesthesia is well known. The present study was undertaken to quantitate the effects of hydrostatic pressure and anesthetics upon the phase-transition temperature of dipalmitoyl phosphatidylcholine vesicles. The drugs used to anesthetize the phospholipid vesicles included an inhalation anesthetic, halothane, a dissociable local anesthetic, lidocaine and an undissociable local anesthetic, benzyl alcohol. All anesthetics decreased the phase-transition temperature dose-dependently. In the case of lidocaine, the depression was pH dependent and only uncharged molecules were effective. The application of hydrostatic pressure increased the phase-transition temperature both in the presence and the absence of anesthetics. The temperature-pressure relationship was linear over the entire pressure range studied up to 340 bars. Through the use of Clapeyron-Clausius equation, the volume change accompanying the phase-transition of the membrane was calculated to be 27.0 cm3/mol. Although the anesthetics decreased the phase-transition temperature, the molar volume change accompanying the phase-transition was not altered. The anesthetics displaced the temperature-pressure lines parallel to each other. The mole fraction of the anesthetics in the liquid crystalline membrane, calculated from the van't Hoff equation, was independent of pressure. This implies that pressure does not displace the anesthetics from the liquid membrane, and the partition of these agents remains constant. The volume change of the anesthetized phospholipid membranes is entirely dependent upon the phase-transition and not on the space occupied by the anesthetics.     

Four-α-Helix Bundle with Designed Anesthetic Binding Pockets. Part II: Halothane Effects on Structure and Dynamics

As a model of the protein targets for volatile anesthetics, the dimeric four-α-helix bundle, (Aα₂-L1M/L38M)₂, was designed to contain a long hydrophobic core, enclosed by four amphipathic α-helices, for specific anesthetic binding. The structural and dynamical analyses of (Aα₂-L1M/L38M)₂ in the absence of anesthetics (another study) showed a highly dynamic antiparallel dimer with an asymmetric arrangement of the four helices and a lateral accessing pathway from the aqueous phase to the hydrophobic core. In this study, we determined the high-resolution NMR structure of (Aα₂-L1M/L38M)₂ in the presence of halothane, a clinically used volatile anesthetic. The high-solution NMR structure, with a backbone root mean-square deviation of 1.72 Å (2JST), and the NMR binding measurements revealed that the primary halothane binding site is located between two side-chains of W15 from each monomer, different from the initially designed anesthetic binding sites. Hydrophobic interactions with residues A44 and L18 also contribute to stabilizing the bound halothane. Whereas halothane produces minor changes in the monomer structure, the quaternary arrangement of the dimer is shifted by about half a helical turn and twists relative to each other, which leads to the closure of the lateral access pathway to the hydrophobic core. Quantitative dynamics analyses, including Modelfree analysis of the relaxation data and the Carr-Purcell-Meiboom-Gill transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange both near and remote from the binding site. Our results revealed a novel mechanism of an induced fit between anesthetic molecule and its protein target, with the direct consequence of protein dynamics changing on a global rather than a local scale. This mechanism may be universal to anesthetic action on neuronal proteins.     

The in vivo neurochemistry of the brain during general anesthesia

Anesthesia describes a complex state composed of immobility, amnesia, hypnosis (sleep or loss of consciousness), analgesia, and muscle relaxation. Bottom-up approaches explain anesthesia by an interaction of the anesthetic with receptor proteins in the brain, whereas top-down approaches consider predominantly cortical and thalamic network activity and connectivity. Both approaches have a number of explanatory gaps and as yet no unifying view has emerged. In addition to a direct interaction with primary target receptor proteins, general anesthetics have massive effects on neurotransmitter activity in the brain. They can change basal transmitter levels by interacting with neuronal activity, transmitter synthesis, release, reuptake and metabolism. By that way, they can affect a great number of neurotransmitter systems and receptors. Here, we review how different general anesthetics affect extracellular activity of neurotransmitters in the brain during induction, maintenance, and emergence from anesthesia and which functional consequences this may have. Commonalities and differences between different groups of anesthetics in their action on neurotransmitter activity are discussed. We also review how general anesthetics affect the response dynamics of the neurotransmitter systems after sensory stimulation. More than 30 years of research have now yielded a complex picture of the effects of general anesthetics on brain neurotransmitter basal activity and response dynamics. It is suggested that analyzing the effects on neurotransmitter activity is the logical next step after protein interactions in a bottom-up analysis of anesthetic action in the brain on the way to a unifying view of anesthesia.     

Anesthetic inhibition of firefly luciferase, a protein model for general anesthesia, does not exhibit pressure reversal.

The surprising observation that pressures of the order of 150 atmospheres can restore consciousness to an anesthetized animal has long been central to theories of the molecular mechanisms underlying general anesthesia. We have constructed a high-pressure gas chamber to test for "pressure reversal" of the best available protein model of general anesthetic target sites: the pure enzyme firefly luciferase, which accounts extremely well for animal potencies (over a 100,000-fold range). We found no significant pressure reversal for a variety of anesthetics of differing size and polarity. It thus appears that either firefly luciferase is not an adequate model for general anesthetic target sites or that pressure and anesthetics act at different molecular sites in the central nervous system.     

Cardiopulmonary,hematological, serum biochemical and behavioral effects of sevoflurane compared with isoflurane or halothane in spontaneously ventilating goats

This study compares cardiopulmonary, hematological, serum biochemical and behavioral effects of sevoflurane, isoflurane or halothane anesthesia in spontaneously breathing, conventionally medicated goats. Six male adult goats were anesthetized repeatedly at 2-week intervals with three anesthetics. Goats were administered atropine (0.1 mg/kg) intramuscularly, and 10 min later, induced to anesthesia by an intravenous infusion of thiopental (mean 14.3 mg/kg). After intubation, goats were anesthetized with halothane, isoflurane or sevoflurane in oxygen and maintained at surgical depth of anesthesia for 3 h. Recovery from anesthesia with sevoflurane was more rapid than that with isoflurane or halothane. Time-related hypercapnia and acidosis were observed during halothane anesthesia, but not observed during sevoflurane or isoflurane anesthesia. Both hypercapnia and acidosis during sevoflurane anesthesia did not differ from isoflurane anesthesia, but were less during halothane anesthesia, especially at prolonged maintenance period. There were no significant differences between anesthetics in respiration and heart rates, arterial pressures, hematological and serum biochemical values. It was concluded that sevoflurane is an effective inhalant for use in goats showing the most rapid recovery from anesthesia, and that cardiopulmonary effects of sevoflurane are similar to isoflurane than halothane.     

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K_D) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.