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Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 [.proportional]M and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons).

From the discovery of anesthesia in 1846 until the 1960s, anesthetic investigations focused largely on the management of clinical problems. The 1960s saw increasing attention to the dose-related effects of anesthesia on the vital processes of the body. Investigators interested in anesthesia developed tools for defining anesthetic kinetics (what the body does to anesthetics) and comparative pharmacodynamic effects (the relative effects of what anesthetics do to the body). In 1968, the Canadian Journal of Anaesthesia published an article by Tsutomu Oyama (1923-2008) et al. entitled ‘‘Effects of halothane anaesthesia and surgery on adrenocortical function in man’’, which is an exemplar presaging the expansion of pharmacodynamic studies. Before the 1960s, relatively little was known of the effects of anesthetics on the body, in this case, specifically the release of free corticosteroids, such as, 17hydroxycorticosteroids (17-OHCS), especially in humans. Oyama opened a new window.

Background Temporary, antegrade amnesia is one of the core desirable endpoints of general anesthesia. Multiple lines of evidence support a role for the hippocampal θ rhythm, a synchronized rhythmic oscillation of field potentials at 4-12 Hz, in memory formation. Previous studies have revealed a disruption of the θ rhythm at surgical levels of anesthesia. We hypothesized that θ-rhythm modulation would also occur at subhypnotic but amnestic concentrations. Therefore, we examined the effect of three inhaled agents on properties of the θ rhythm considered critical for the formation of hippocampus-dependent memories. Methods We studied the effects of halothane and nitrous oxide, two agents known to modulate different molecular targets (GABAergic [γ-aminobutyric acid] vs. non-GABAergic, respectively) and isoflurane (GABAergic and non-GABAergic targets) on fear-conditioned learning and θ oscillations in freely behaving rats. Results All three anesthetics slowed θ peak frequency in proportion to their inhibition of fear conditioning (by 1, 0.7, and 0.5 Hz for 0.32% isoflurane, 60% N2O, and 0.24% halothane, respectively). Anesthetics inconsistently affected other characteristics of θ oscillations. Conclusions At subhypnotic amnestic concentrations, θ-oscillation frequency was the parameter most consistently affected by these three anesthetics. These results are consistent with the hypothesis that modulation of the θ rhythm contributes to anesthetic-induced amnesia.

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABA(A) receptors (GABA(A)-Rs) in a manner that makes them plausible targets. We asked whether GABA(A)-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABA(A)-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABA(A)-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC(50) for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N(2) generation knockins. This effect was not observed at the N(4) generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC(50)) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit.

The molecular site of action for volatile anesthetics remains unknown despite many years of study. Members of the K2P potassium channel family, whose currents are potentiated by volatile anesthetics have emerged as possible anesthetic targets. In fact, a mouse model in which the gene for TREK-1 (KCNK2) has been inactivated shows resistance to volatile anesthetics. In this study we tested whether inactivation of another member of this ion channel family, KCNK7, in a knockout mouse displayed altered sensitivity to the anesthetizing effect of volatile anesthetics. KCNK7 knockout mice were produced by standard gene inactivation methods. Heterozygous breeding pairs produced animals that were homozygous, heterozygous or wildtype for the inactivated gene. Knockout animals were tested for movement in response to noxious stimulus (tail clamp) under varying concentrations of isoflurane, sevoflurane and desflurane to define the minimum alveolar concentration (MAC) preventing movement. Mice homozygous for inactivated KCNK7 were viable and indistinguishable in weight, general development and behavior from heterozygotes or wildtype littermates. Knockout mice (KCNK7 −/−) displayed no difference in MAC for the three volatile anesthetics compared to heterozygous (+/−) or wildtype (+/+) littermates. Because inactivation of KCNK7 does not alter MAC, KCNK7 may play only a minor role in normal CNS function or may have had its function compensated for by other inhibitory mechanisms. Additional studies with transgenic animals will help define the overall role of the K2P channels in normal neurophysiology and in volatile anesthetic mechanisms.

Results from several studies point to sodium channels as potential mediators of the immobility produced by inhaled anesthetics. We hypothesized that the intrathecal administration of veratridine, a drug that enhances the activity or effect of sodium channels, should increase MAC.We measured the change in isoflurane MAC caused by intrathecal infusion of various concentrations of veratridine into the lumbothoracic subarachnoid space of rats. We compared these result with those obtained from intracerebroventricular infusion.As predicted, intrathecal infusion of veratridine increased MAC. The greatest infused concentration (25 microM) also produced neuronal injury in the hindlimbs of two rats and decreased the peak effect on MAC. A concentration of 1.6 microM produced the largest (21%) increase in MAC. Intraventricular infusion of 1.6 and 6.4 microM veratridine did not alter MAC. Rats given 25 microM died.Intrathecal administration of veratradine increases MAC of isoflurane, a finding consistent with a role for sodium channels as potential mediators of the immobility produced by inhaled anesthetics.

A paradox arises from present information concerning the mechanism(s) by which inhaled anesthetics produce immobility in the face of noxious stimulation. Several findings, such as additivity, suggest a common site at which inhaled anesthetics act to produce immobility. However, two decades of focused investigation have not identified a ligand- or voltage-gated channel that alone is sufficient to mediate immobility. Indeed, most putative targets provide minimal or no mediation. For example, opioid, 5-HT3, gamma-aminobutyric acid type A and glutamate receptors, and potassium and calcium channels appear to be irrelevant or play only minor roles. Furthermore, no combination of actions on ligand- or voltage-gated channels seems sufficient. A few plausible targets (e.g., sodium channels) merit further study, but there remains the possibility that immobilization results from a nonspecific mechanism.

BACKGROUND:The purpose of this study was to determine whether pairs of compounds, including general anesthetics, could simultaneously modulate receptor function in a synergistic manner, thus demonstrating the existence of multiple intraprotein anesthetic binding sites.METHODS:Using standard electrop

BACKGROUND: Inhaled anesthetics have been postulated to act at multiple receptors, with modest action at each site summing to produce immobility to noxious stimulation. Recent experimental results affirm prior findings that inhaled anesthetics interact additively. Synergy implies multiple sites of action by definition. In this essay, we explore the converse: does additivity imply a single site of action? METHODS: The interaction of one versus two ligands competing for the same binding site at a receptor was explored using the law of mass action. Circuits were then constructed to investigate how the potency of drugs and the steepness of the concentration versus response relationship is amplified by the arrangement of suppressors into serial circuits, and enhancers into parallel circuits. Assemblies of suppressor and enhancer circuits into signal processing units were then explored to investigate the constraints signal processing units impose on additive interactions. Lastly, the relationship between synergy, additivity, and fractional receptor occupancy was explored to understand the constraints imposed by additivity. RESULTS: Drugs that compete for a single receptor, and that similarly affect the receptor, must be additive in their effects. Receptors that bind suppressors in serial circuits, or enhancers in parallel circuits, increase the apparent potency of the drugs and the steepness of the concentration versus response relationship. When assemblies of suppressor and enhancer circuits are arranged into signal processing units, the interactions may be additive or synergistic. The primary determinant is the relationship between the concentration of drug associated with the effect of interest and the concentration associated with 50% receptor occupancy, kd. Effects mediated by very low concentrations are more likely to be additive. Similarly, inhaled anesthetics that act at separate sites are unlikely to exhibit additive interactions if anesthetic drug effect occurs at concentrations at or above 50% receptor occupancy. However, if anesthetic drug effect occurs at very low levels of receptor occupancy, then additivity is expected even among anesthetics acting on different receptors. CONCLUSIONS: Additivity among drugs acting on different receptors is only likely if the concentrations responsible for the drug effect of interest are well below the concentration associated with 50% receptor occupancy. (Anesth Analg 2008;107:507‐24)

BACKGROUND: Drug interactions may reveal mechanisms of drug action: additive interactions suggest a common site of action, and synergistic interactions suggest different sites of action. We applied this reasoning in a review of published data on anesthetic drug interactions for the end-points of hypnosis and immobility. METHODS: We searched Medline for all manuscripts listing propofol, etomidate, methohexital, thiopental, midazolam, diazepam, ketamine, dexmedetomidine, clonidine, morphine, fentanyl, sufentanil, alfentanil, remifentanil, droperidol, metoclopramide, lidocaine, halothane, enflurane, isoflurane, sevoflurane, desflurane, N2O, and Xe that contained terms suggesting interaction: interaction, additive, additivity, synergy, synergism, synergistic, antagonism, antagonistic, isobologram, or isobolographic. When available, data were reanalyzed using fraction analysis or response surface analysis. RESULTS: Between drug classes, most interactions were synergistic. The major exception was ketamine, which typically interacted in either an additive or infra-additive (antagonistic) manner. Inhaled anesthetics typically showed synergy with IV anesthetics, but were additive or, in the case of nitrous oxide and isoflurane, possibly infra-additive, with each other. CONCLUSIONS: Except for ketamine, IV anesthetics acting at different sites usually demonstrated synergy. Inhaled anesthetics usually demonstrated synergy with IV anesthetics, but no pair of inhaled anesthetics interacted synergistically. (Anesth Analg 2008;107:494‐506)

BACKGROUND Inhaled anesthetics may affect proteins at the interface between membrane lipids and the surrounding aqueous phase. The underlying solution chemistry is not known. Because the hydrophobicity of nonpolar protein components importantly influences their conformation, we tested the hypothesis that isoflurane affects the solubility of two nonpolar compounds, methane and toluene, in saline. METHODS Using a serial dilution technique, we determined the saline:gas partition coefficients (PCs) of methane and toluene at 37 degrees C in the absence of isoflurane and in the presence of approximately 1%, 5%, and 15% isoflurane. We also measured the effect on the vapor pressure of benzene produced by saturating benzene with either cyclopropane or chloroethane, anesthetics used in a previous study to demonstrate that their equilibration with benzene decreased the solubility of benzene in water. RESULTS Clinically relevant concentrations of isoflurane (1% and 5%) did not affect the saline:gas PC of methane and toluene, but 15%-20% isoflurane increased the PC of methane (P < 0.05) but not toluene. Saturating benzene with cyclopropane or chloroethane, decreased the vapor pressure of benzene in proportion to the amount of anesthetic dissolved in the benzene. CONCLUSION Isoflurane has a weak antihydrophobic effect at concentrations far above the clinically relevant range, and this effect is unlikely to explain how anesthetics act. A previous study, which found that cyclopropane and chloroethane decreased the solubility of benzene in water, probably erred in its conclusion that these anesthetics interfered with the interaction of benzene and water. Instead, the anesthetics simply decreased the vapor pressure of benzene, doing so in accordance with Raoult's Law.

Windup is a progressive increase in response of dorsal horn neurons to repetitive C-fiber stimulation that may underlie temporal summation of pain. We investigated the frequency- and intensity-dependency of windup, and the effects of isoflurane and N-methyl-d-aspartate (NMDA) receptor blockade, to determine if they parallel the influence of temporal and spatial summation of noxious stimuli on anesthetic requirements.We recorded responses of rat sacral dorsal horn neurons to 20-s trains of electrical tail stimulation at different frequencies (0.3-10 Hz) and intensities (0.8-5 x stimulus threshold) during delivery of 0.7 to 1.3 minimum alveolar anesthetic concentration isoflurane. Summed responses (area under the curve [AUC] windup), initial response, absolute windup (AUC minus 20 times the initial response), and slope of windup were quantified.Increases in stimulus intensity and frequency progressively increased AUC windup (P0.01 for both) and correlated with isoflurane concentrations required for immobility (R2 = 0.98 and 0.97, respectively). Increasing the isoflurane concentration significantly suppressed each measure of windup elicited by low-intensity and low-frequency, but not high-intensity and high-frequency stimulus trains. The initial response magnitude significantly correlated with slope of windup across stimulus intensities and isoflurane concentrations. The NMDA receptor antagonist MK801 significantly reduced windup (to 53%; P0.05) at 1 Hz.Windup of dorsal horn neurons at low stimulus intensities and frequencies increases isoflurane requirements for immobility via a NMDA receptor-dependent mechanism. At high stimulus intensities and frequencies, windup was resistant to isoflurane consistent with larger anesthetic requirements for immobility.

BACKGROUND: Previous studies demonstrated that MAC (the minimum alveolar concentration of an inhaled anesthetic that produces immobility in 50% of subjects exposed to noxious stimulation) for halothane directly correlates with the central nervous system concentration of Na + . However, those studies globally altered Na + concentrations, and thus did not distinguish effects on the spinal cord from cerebral effects. This is an important distinction because the cord appears to be the primary site for mediation of the immobility produced by inhaled anesthetics. Accordingly, in the present study, we examined the effect of altering intrathecal versus intracerebroventricular concentrations of Na + on MAC. METHODS: In rats prepared with chronic indwelling catheters or stylets, we infused solutions deficient in Na + and with an excess of Na + into the lumbar subarachnoid and intracerebroventricular spaces and measured MAC for isoflurane before, during, and after infusion. RESULTS: MAC of isoflurane correlated directly with concentrations of Na + infused intrathecally but did not correlate with concentrations infused intracerebroventricularly. CONCLUSION: The results are consistent with a mediation or modulation of MAC by Na + channels. These might include voltage-gated or ligand-gated channels or other Na-sensitive targets (e.g., pumps, transporters, exchangers).

The minimum alveolar concentration (MAC) of isoflurane is a quantitative trait because it varies continuously in a population. The location on the genome of genes or other genetic elements controlling quantiative traits is called quantitative trait loci (QTLs). In this study we sought to detect a quantitative trait locus underlying isoflurane MAC in mice.To accomplish this, two inbred mouse strains differing in isoflurane MAC, the C57BL/6J and LP/J mouse strains, were bred through two generations to produce genetic recombination. These animals were genotyped for microsatellite markers. We also applied an independent, computational method for identifying QTL-regulating differences in isoflurane MAC. In this approach, the isoflurane MAC was measured in a panel of 19 inbred strains, and computationally searched for genomic intervals where the pattern of genetic variation, based on single nucleotide polymorphisms, correlated with the differences in isoflurane MAC among inbred strains.Both methods of genetic analysis identified a QTL for isoflurane MAC that was located on the proximal part of mouse chromosome 7.

INTRODUCTION Intravenous (IV) fluid bags made of polyvinyl chloride (PVC) often contain the plasticizer di(2-ethylhexyl) phthalate (DEHP) to make the PVC flexible. Phthalate esters have been reported to inhibit neuronal nicotinic acetylcholine receptors, which are sensitive to many inhaled anesthetics. This raises the possibility that DEHP might modulate the function of other cys-loop receptors, such as gamma-amino butyric acid type A (GABA(A)) and glycine receptors, and that DEHP-plasticized PVC might interfere with electrophysiologic studies of anesthetic mechanisms on those receptors. METHODS alpha(1)beta(2) GABA(A) and alpha(1) glycine receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. We then measured the effect of buffers from IV bags containing DEHP-plasticized PVC, and of buffers saturated with DEHP, on agonist-induced currents. RESULTS Agonist-induced currents from glycine receptors were enhanced by buffers from IV bags containing DEHP-plasticized PVC by 291.9% +/- 84.5% (mean +/- se) and from saturated solutions of DEHP by 70.8% +/- 16.7%. Agonist-induced currents from alpha(1)beta(2) GABA(A) receptors were inhibited by buffers from IV bags containing DEHP-plasticized PVC by 19.3% +/- 3.2% and by 31.7% +/- 7.0% from buffers saturated with DEHP. CONCLUSIONS The plasticizer DEHP modulates the function of both GABA(A) and glycine receptors. DEHP contamination can confound the results of electrophysiologic studies of anesthetic mechanisms on these receptors if DEHP-plasticized PVC is present in the experimental apparatus.

Previous studies have found that the local anesthetic/sodium channel blocker lidocaine decreased MAC by maximum amounts approximately equal to the decreases produced by dizocilpine (MK-801), a N-methyl-d-aspartate (NMDA) receptor antagonist. Blockade of sodium channels by inhaled anesthetics has been suggested as a possible cause for impairment of transmission through NMDA receptors. We postulated that the net effect of lidocaine and MK-801 on MAC would be the same, albeit by affecting NMDA neurotransmission at different points.We measured the effect of various lidocaine infusions on the MAC of cyclopropane, halothane, isoflurane, and o-difluorobenzene in rats. We also measured the effect of concurrent lidocaine-MK-801 infusion on the MAC of isoflurane and o-difluorobenzene.Our data contradicted our predictions. (a) We found no limit to the effect of lidocaine infusion, in some cases finding that lidocaine, alone, produced immobility; (b) lidocaine infusion did not decrease the MAC of o-difluorobenzene differently from the MAC of other inhaled anesthetics; and (c) the addition of MK-801 equally affected the decrease in MAC produced by lidocaine infusion for isoflurane versus o-difluorobenzene.Lidocaine does not primarily decrease MAC by decreasing the release of glutamate from nerve terminals.

BACKGROUND: The search for new potent inhaled anesthetics has slowed, in large part because of the excellence of the two most recent additions, desflurane and sevoflurane. Nonetheless, neither desflurane nor sevoflurane are ideal anesthetics, desflurane causing cardiorespiratory stimulation, and sevoflurane having a slower (albeit rapid) recovery from anesthesia. Sevoflurane also can produce convulsions and postoperative agitation. METHODS AND RESULTS: In the present report, we describe the physical and anesthetic properties of 31 cyclic ethers halogenated solely with fluorine. Although several produced anesthesia, none had solubilities that would make them better than sevoflurane. The remaining ethers were unstable or produced obvious central nervous system irritation, including convulsions. CONCLUSIONS: We find that none of these cyclic ethers appear to provide advantages over desflurane or sevoflurane.

BACKGROUND: Because of the logistical obstacles to measurement under hyperbaric conditions, the effect of nitrous oxide (N 2 O) alone on spinal neuronal responses has not been tested. We hypothesized that, like other inhaled anesthetics, N 2 O would depress spinal neuronal responses to noxious stimulation. METHODS: The lumbar spinal cord was exposed in rats anesthetized with isoflurane. Mechanically ventilated rats were placed into a hyperbaric chamber and needle electrodes were inserted into the hindpaws. Isoflurane administration was discontinued and anesthesia converted to N 2 O by pressurizing the chamber with N 2 O. A microelectrode was inserted into the lumbar cord using computer-controlled motors and a hydraulic microdrive. Neuronal responses to electrical stimulation of the hindpaw were sought at 1.5, 2, and 2.5 atm N 2 O (0.8-1.3 minimum alveolar concentration). RESULTS: Increasing N 2 O partial pressures variably affected neuronal responses to a 2 s 100-Hz electrical stimulus. Neuronal depth and neuronal response were correlated, with superficial neurons tending to be facilitated, while deeper neurons were depressed; (overall responses were 1331 ± 408, 1594 ± 383, and 1578 ± 500 impulses/min at 1.5, 2, and 2.5 atm N 2 O, respectively; mean, standard error). N 2 O did not affect neuronal responses to a repetitive "windup" stimulus. Infusion of the N-methyl-D-aspartate blocker MK-801 into separate rats increased the neuronal response to the 100-Hz stimulus (from 781 ± 216 to 1352 ± 269 impulses/min, P < 0.05). CONCLUSIONS: N 2 O facilitated superficial spinal neuronal responses to noxious stimulation while depressing deeper neurons. These results suggest that anesthetic partial pressures of N 2 O have divergent effects on spinal neuronal responses to noxious stimulation, the specific responses depending on the depth of the spinal neurons.

Despite the known capacity of hypothermia to increase anesthetic potency (decrease the partial pressure required to produce anesthesia), many in vitro studies examine the effects of ethanol and other anesthetics in oocytes or isolated neurons at room temperature. We tested whether, as predicted for potent inhaled anesthetics, a proportionate increase in solubility with hypothermia matched a decrease in ethanol minimum alveolar concentration (MAC), and thereby made the use of a single anesthetic concentration appropriate regardless of temperature. We determined ethanol MAC in normothermic (37.3 degrees C) and hypothermic (28.5 degrees C) rats, and, at the two temperatures, also determined ethanol solubilities in olive oil and saline. Ethanol MAC decreased, while olive oil/gas and saline/gas partition coefficients increased. However, the increase in the saline/gas partition coefficient did not match the decrease in MAC, and thus the aqueous-phase partial pressure producing absence of movement in 50% of rats (EC50) values for ethanol decreased by 17%. Although this decrease is not large, it may be important for comparative estimates of the in vitro effects of ethanol at different temperatures.

Most studies of chirality in inhaled anesthetic action have used the enantiomers of isoflurane. These enantiomers are expensive and scarce, which limits studies, such as the preliminary identification of molecular targets of anesthetic action, that can be performed with these isomers. We hypothesized that secondary alcohols (i.e., compounds having a -CH 2 -CHOH-CH 3 group) that are experimental anesthetics would show enantioselectivity. To test this hypothesis, we determined the minimum alveolar anesthetic concentration (MAC) of the enantiomers of the homologous series of 2-alcohols from 2-butanol to 2-heptanol in rats. Because these alcohols are partially metabolized to 2-ketones during the course of study (i.e., having a -CH 2 -CO-CH 3 group), we independently measured the MAC of the 2-ketones. Assuming additivity of MAC of the ketones with the alcohols, we corrected for the anesthetic effect of the ketones in rats to determine the MAC of the alcohols. We found that the 2-butanol and 2-pentanol isomers were enantioselective. S-(+)-2-butanol had a MAC that was 17% larger than for the R-(-)-enantiomer, whereas S-(+)-2-pentanol had a MAC that was 38% larger than the R-(-)-enantiomer. No stereoselectivity was observed for 2-hexanol and 2-heptanol. These findings may permit studies of chirality in anesthesia, particularly in in vitro systems where metabolism does not occur, using inexpensive volatile compounds.

Chirality has been proposed as a means for distinguishing relevant from irrelevant molecular targets of action, but the sensitivity and specificity of this test is unknown for volatile anesthetics. We applied enantiomers of two chiral anesthetic alcohols (2-butanol and 2-pentanol) that are enantioselective for the minimum alveolar concentration (MAC) preventing movement in 50% of animals and one (2-hexanol) that was not to frog oocytes. Each oocyte expressed one of three anesthetic-sensitive ion channels: a Twik-related-spinal cord K+ (TRESK) channel, a gamma-amino butyric acid type A (GABA(A)) receptor and an N-methyl-d-aspartate (NMDA) receptor. Using voltage-clamp techniques, we found that 2-butanol was not enantioselective for any channel (e.g., 16 mM 2-butanol R(-) and S(-) enantiomers decreased current through an NMDA receptors by 44% +/- 3% [mean +/- se] and 37% +/- 4%, respectively); 2-pentanol was enantioselective for one channel (the GABA(A) receptor, the enantiomers increasing current by 277% +/- 20% and 141% +/- 30%); 2-hexanol was enantioselective for both GABA(A) and NMDA receptors (e.g., decreasing current through the NMDA receptor by 19% +/- 3% and 43% +/- 5%). We calculated the sensitivity and specificity of chirality as a test of anesthetic relevance under two scenarios: 1) all three channels were relevant mediators of MAC and 2) no channel was a mediator of MAC. These sensitivities and specificities were poor because there is no consistent correspondence between receptor and whole animal results. We recommend that enantioselectivity not be used as a test of relevance for inhaled anesthetic targets.

We hypothesized that N-methyl-d-aspartate (NMDA) receptors mediate some or all of the capacity of inhaled anesthetics to prevent movement in the face of noxious stimulation, and that this capacity to prevent movement correlates directly with the in vitro capacity of such anesthetics to block the NMDA receptor. To test this hypothesis, we measured the effect of IV infusion of the NMDA blockers dizocilpine (MK-801) and (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid (CPP) to decrease the MAC (the minimum alveolar concentration of anesthetic that prevents movement in 50% of subjects given a noxious stimulation) of 8 conventional anesthetics (cyclopropane, desflurane, enflurane, halothane, isoflurane, nitrous oxide, sevoflurane, and xenon) and 8 aromatic compounds (benzene, fluorobenzene, o-difluorobenzene, p-difluorobenzene, 1,2,4-trifluorobenzene, 1,3,5-trifluorobenzene, pentafluorobenzene, and hexafluorobenzene) and, for comparison, etomidate. We postulated that MK-801 or CPP infusions would decrease MAC in inverse proportion to the in vitro capacity of these anesthetics to block the NMDA receptor. This notion proved correct for the aromatic inhaled anesthetics, but not for the conventional anesthetics. At the greatest infusion of MK-801 (32 microg x kg(-1) x min(-1)) the MACs of conventional anesthetics decreased by 59.4 +/- 3.4% (mean +/- sd) and at 8 microg x kg(-1) x min(-1) by 45.5 +/- 4.2%, a decrease not significantly different from a 51.4 +/- 19.0% decrease produced in the EC50 for etomidate, an anesthetic that acts solely by enhancing gamma-amino butyric acid (GABA) receptors. We conclude that some aromatic anesthetics may produce immobility in the face of noxious stimulation by blocking the action of glutamate on NMDA receptors but that conventional inhaled anesthetics do not.

Several previous studies concluded that opioid receptors do not mediate the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation because administration of naloxone (a nonspecific opioid receptor antagonist) does not increase the minimum alveolar anesthetic concentration (MAC) of inhaled anesthetic that produces immobility in 50% of subjects given a noxious stimulation. In contrast, a recent study found that 0.1 mg/kg naloxone given intraperitoneally increased sevoflurane MAC in mice by 18% (P < 0.01). We repeated the recent study with sevoflurane in the same strain of mice, administering nothing (control), 0.1 mg/kg, and 1.0 mg/kg of naloxone. Our study differed in that we also tested a parallel group given saline rather than naloxone. We were blinded to drug administration. MAC decreased 4.8% +/- 11.0% (mean+/- sd) and 2.4% +/- 12.5% with the first and second administrations of saline. Similarly, MAC decreased 4.7% +/- 7.1% and 5.5% +/- 10.0% with the administration of 0.1 mg/kg and 1.0 mg/kg of naloxone. We do not find that naloxone increases MAC. Opioid receptors do not underlie a portion of the capacity of inhaled anesthetics to produce immobility.

The Meyer-Overton hypothesis predicts that anesthetic potency correlates inversely with lipophilicity; e.g., MAC times the olive oil/gas partition coefficient equals a constant of approximately 1.82 +/- 0.56 atm (mean +/- sd) for conventional inhaled anesthetics. MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. In contrast to conventional inhaled anesthetics, MAC times the olive oil/gas partition coefficient for normal alcohols from methanol through octanol equals a constant one tenth as large as that for conventional inhaled anesthetics. The alcohol (C-OH) group causes a great affinity of alcohols to water, and the C-OH may tether the alcohol at the hydrophobic-hydrophilic interface where anesthetics are thought to act. We hypothesized that the position of the C-OH group determined potency, perhaps by governing the maximum extent to which the acyl portion of the molecule might extend into a hydrophobic phase. Using the same reasoning, we added studies of ketones with similar numbers of carbon atoms between the C=O group and the terminal methyl group. The results for both alcohols and ketones showed the predicted correlation, but the correlation was no better than that with carbon chain length regardless of the placement of the oxygen. The oil/gas partition coefficient predicted potency as well as, or better than, either chain length or oxygen placement. Hydrophilicity, as indicated by the saline/gas partition coefficient, also seemed to influence potency.

Context-sensitive decrement times for inhaled anesthetics connect two values: a) the duration of anesthesia (nominally at a constant alveolar concentration)-the "context" and b) the time to decrease the alveolar or vital tissue (e.g., brain, heart, kidney, and liver, collectively called the vessel-rich group of tissues) concentration by some fractional "decrement" of the starting concentration. Increasing duration of anesthesia increases the time to a given decrement in a nonlinear manner that may considerably delay recovery. In the present report we use a commercially available simulation program (Gas Man) to confirm and enlarge on these concepts. In this simulation, increasing duration of anesthesia can markedly delay complete awakening for isoflurane. Increasing anesthesia duration imposes considerably less delay in awakening from sevoflurane compared with isoflurane. For desflurane, only prolonged anesthesia or decrements of 95% and more should delay awakening from anesthesia. These changes are shown to be the result of the relative solubility of each anesthetic in blood and tissue. An increase in cardiac output is also shown to delay awakening.

Rarely, fire and patient injury result from the degradation of sevoflurane by desiccated Baralyme. The present investigation sought to determine whether high temperatures also arose with sevoflurane use in the presence of desiccated soda lime. We desiccated soda lime by directing a 10 L/min flow of oxygen through fresh absorbent. Using 1140 +/- 30 g (mean +/- sd) of this desiccated absorbent, we filled a single standard absorber canister placed in a standard anesthetic circuit to which we directed a 6 L/min flow of oxygen containing 1.5 minimum alveolar concentration (MAC) desflurane or sevoflurane, or 3.0 MAC desflurane, isoflurane, or sevoflurane (with and without concurrent delivery of 200 mL/min carbon dioxide). In an additional test, 2 canisters (rather than a single canister) containing desiccated absorbent were used and 3.0 MAC sevoflurane was applied. A 3-L reservoir bag served as a surrogate lung, and we ventilated this lung with a minute ventilation of 10 L/min. With desflurane at 1.5 MAC or 3.0 MAC or isoflurane at 3.0 MAC temperatures increased in 20 to 40 min to a peak of 30 degrees C to 45 degrees C and then declined. With 1.5 or 3.0 MAC sevoflurane, temperatures increased to approximately 90 degrees C, after which temperatures declined. Concurrent delivery of carbon dioxide and sevoflurane did not increase the peak temperatures reached. The use of 2 canisters increased the duration but not the peak of increased temperature reached with 3.0 MAC sevoflurane. No fires resulted from degradation of any anesthetic.

Combinations of GluR5-GluR7, KA1, and KA2 subunits form kainate receptors, a subtype of excitatory ionotropic glutamate receptors. Isoflurane enhances the action of kainate receptors comprising GluR6 subunits expressed in oocytes. To test whether alterations of the GluR6 subunit gene affect the actions of inhaled anesthetics in vivo, we measured the minimum alveolar concentration of desflurane, isoflurane, and halothane in mice lacking the kainate receptor subunit GluR6 (GluR6 knockout mice) and mice with a dominant negative glutamine/arginine (Q/R) editing mutation in membrane domain 2 of the GluR6 receptor (GluR6 editing mutants), which increases the calcium permeability of kainate receptors containing GluR6Q. We also measured the capacity of isoflurane to interfere with Pavlovian fear conditioning to a tone and to context. Absence of the GluR6 subunit did not change the minimum alveolar concentration of isoflurane, desflurane, or halothane. Possibly, kainate receptors assembled from the remaining kainate receptor subunits compensate for the absent subunits and thereby produce a normal minimum alveolar concentration. A Q/R mutation that dominantly affects kainate receptors containing the GluR6 subunit in mice increased isoflurane minimum alveolar concentration (by 12%; P < 0.01), decreased desflurane minimum alveolar concentration (by 18%; P < 0.001), and did not change halothane minimum alveolar concentration (P = 0.25). These data may indicate that kainate receptors containing GluR6Q subunits differently modulate, directly or indirectly, the mechanism by which inhaled anesthetics cause immobility. The mutations of GluR6 that were studied did not affect the capacity of isoflurane to interfere with fear conditioning.

A photoactivable diazirine derivative of etomidate, azietomidate, shares many actions of etomidate, including a capacity to abolish the righting reflexes in tadpoles and enhance gamma-aminobutyric acid (GABA)-induced currents. Azietomidate's usefulness in studies of mechanisms of anesthesia depends on the assumption that it shares a site of action with etomidate. Mice bearing an N265M beta3 subunit point mutation in GABA(A) receptors have a markedly decreased sensitivity to loss of righting reflexes induced by etomidate over a range of doses. Accordingly, in the present study we measured the time to recovery of righting reflexes of wild type and mutant mice as a function of dose given as an IV bolus. Analysis of the data for azietomidate yielded mean times to recovery of righting reflexes at a dose of 7.5 mg/kg of 10.0 +/- 0.9 min and 3.0 +/- 1.6 min for wild type and mutant mice, respectively (mean +/- sd). A similar analysis for etomidate yielded mean times to recovery of righting reflexes at a dose of 7.5 mg/kg of 12.0 +/- 1.3 min and 4.0 +/- 0.7 min for wild type and mutant mice respectively. Thus, at this dose a single mutation, N265M on the beta3 subunit of the GABA(A) receptor, approximately halved the time to recovery of righting reflexes for both etomidate and azietomidate (by 7.6 +/- 1.5 min and 7.2 +/- 1.8 min, respectively), emphasizing the contribution of this residue as a determinant of a behavioral response of azietomidate in mice.

Although known for whole brain, values are lacking for solubilities of modern volatile anesthetics in specific brain regions. Some regions should differ from others (e.g., gray matter versus white matter) because they differ in lipid content and because potent inhaled anesthetics are lipophilic. In the present report, we examined this issue in bovine brain, finding that white matter/gas partition coefficients are 1.6 (desflurane) to 2.4 (halothane) times larger than gray matter/gas partition coefficients, with values for isoflurane and sevoflurane lying between these at 1.9. Values for thalamus/gas, hypothalamic area/gas, and hippocampal/gas partition coefficients lie between those for gray and white matter. These data may be useful in defining the parts of the brain involved with return to consciousness during recovery from anesthesia.

Although several mathematical and computer simulations of inhaled anesthetic pharmacokinetics have been devised, their complexity sometimes limits an intuitive appreciation of the interactions produced by the determinants of kinetics. In this essay, we illustrate the factors that govern inhaled anesthetic pharmacokinetics with drawings that consider delivery of anesthetic by ventilation to the lungs and dispersion of the anesthetic to tissue depots by the circulation. The illustrations incorporate the effects of both blood flow and blood solubility as determinants of the extent of dispersion. They incorporate tissue volume and solubility as determinants of the capacity of the tissue depots. Capacity to hold (take up) anesthetic is depicted by areas representing specific tissues, and the extent of anesthetic movement is depicted by the length and breadth of arrows to and from the areas depicting capacity. The illustrations incorporate increasingly important elements to kinetics, such as obesity. Obesity increases the depots available for storage of anesthetic, including anesthetic that reaches fat by intertissue diffusion. Such anesthetic returns to the circulation to delay recovery in healthy and obese patients, particularly with more soluble anesthetics. However, the increased anesthetic in fat occurs at a lower partial pressure and thus might not influence emergence materially. We hope that these illustrations will allow anesthesia practitioners to appreciate the interactions of the factors that govern inhaled anesthetic pharmacokinetics.

Rarely, fire and patient injury have resulted from the degradation of sevoflurane by desiccated carbon dioxide absorbent. Desiccated absorbent also can degrade desflurane and isoflurane, and in the present investigation we sought to determine whether a danger of fire also arose with their use in the presence of desiccated absorbent. Baralyme was desiccated by heating and directing a 10 L/min flow of oxygen through the absorbent. Approximately 1200 g of this desiccated absorbent was used to fill a standard absorber placed in a standard anesthetic circuit to which we directed a 6 L/min flow of oxygen containing 1.5 or 3.0 MAC desflurane, isoflurane, or sevoflurane. A 3-L reservoir bag served as a surrogate lung, and we ventilated this lung with a minute ventilation of 10 L/min. With desflurane or isoflurane, at both 1.5 MAC and 3.0 MAC, temperatures increased in 30 to 70 min to a peak of approximately 100 degrees C and then decreased. With 1.5 MAC sevoflurane (3.0 MAC was not studied), temperatures increased to over 200 degrees C, and in 2 of 5 studies, flames appeared in the anesthetic circuit. In a separate study, we found that concurrent delivery of carbon dioxide and desflurane did not increase peak temperatures. We conclude that the interaction of desflurane or isoflurane with desiccated absorbent is not likely to produce the conflagrations possible with sevoflurane.

Many inhaled anesthetics enhance the effect of the inhibitory neurotransmitter gamma aminobutyric acid (GABA), supporting the view that the GABAA receptor could mediate the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation (i.e., MAC, the minimum alveolar concentration required to suppress movement in response to a noxious stimulus in 50% of subjects). However, only limited in vivo data support the relevance of the GABAA receptor to MAC. In the present study we used two findings to test for the relevance of this receptor to immobilization for isoflurane: 1) differences among anesthetics in their capacity to enhance the response of receptor expression systems to GABA: isoflurane (considerable enhancement), xenon (minimal enhancement), and cyclopropane (minimal enhancement); and 2) studies showing that the spinal cord mediates MAC for isoflurane. If GABAA receptors mediate isoflurane MAC, then their blockade in the spinal cord should increase isoflurane MAC more than cyclopropane or xenon MAC and the MAC increase should be proportional to the in vitro enhancement of the GABAA receptor. To test this thesis, isoflurane, cyclopropane, or xenon MAC was determined in rats during intrathecal infusion of artificial cerebrospinal fluid (aCSF) via chronically implanted catheters. Then MAC was redetermined during infusion of 1 microL/min aCSF containing either 0.6 or 2.4 mg/mL picrotoxin, which noncompetitively blocks GABAA receptors. There was no consistent increase in MAC consequent to increasing the picrotoxin dose from 0.6 to 2.4 microg/min, which suggests that maximal blockade of GABAA receptors in the spinal cord had been achieved. Picrotoxin infusion increased MAC approximately 40% with all anesthetics. This indicates that GABA release in the spinal cord influences anesthetic requirement. However, the increase did not consistently differ among anesthetics and did not correlate with in vitro enhancement of GABAA receptors by these anesthetics. This supports the view that GABAA receptors do not mediate immobilization for isoflurane.

A progressive increase in the response of a nociceptive spinal neuron to repeated electrical C-fiber stimulation reflects a phenomenon called windup. Second order neurons in the dorsal horn, as well as motoneurons, can develop windup. Inhaled anesthetics act primarily in spinal cord to suppress movement induced by noxious stimulation. We hypothesized that halothane would depress neuronal windup in mice at concentrations that also prevented movement. We measured windup in deep dorsal horn neurons in lumbar spinal cord at 0.75 MAC (the minimum alveolar concentration of anesthetic that prevents movement in 50% of subjects in response to noxious stimulation), 0.9 MAC, and 1.1 MAC. The change from 0.75 to 0.9 MAC did not significantly decrease windup (-11+/-22%), but the change from 0.9 to 1.1 MAC decreased windup (-35+/-7%, P0.01). We conclude that halothane depresses neuronal windup in the range that prevents movement, and that the effect on windup might play a role in halothane's immobilizing action.

Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific amino acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., gamma-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and alpha(2)-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-D-aspartate, and sodium) remain credible candidates.

UNLABELLED Many inhaled anesthetics block the in vitro effect of the excitatory neurotransmitter serotonin on the 5-HT2A receptor, supporting the view that this receptor might mediate the capacity of inhaled anesthetics to produce immobility during noxious stimulation (i.e., would underlie MAC, the minimum alveolar concentration required to suppress movement in response to a noxious stimulus in 50% of subjects). In the present investigation in rats, we found that intrathecal administration of the 5HT-2A blocker, ketanserin, can decrease isoflurane MAC. This effect, presumably mediated by blockade of serotonin transmission in the spinal cord, reaches a maximum of 20%-25%. An additional decrease (to 60%) may be obtained by IV infusion of ketanserin, and presumably this decrease results from ketanserin's actions on supraspinal centers. The IV doses of ketanserin that decreased MAC were approximately 100 microg. kg(-1). min(-1) in rats, compared with usual clinical doses of 1.25 microg. kg(-1). min(-1) in humans. These results indicate that 5HT2A receptors are in the neural circuitry influencing isoflurane MAC. These results, together with the blocking action of isoflurane on expressed 5HT2A receptors, strengthen the case for a role for 5HT2A receptors to isoflurane-induced immobility. However, because MAC for isoflurane is predominantly determined in the spinal cord, this result is consistent at most with a minor contribution of these receptors to the immobilizing action of isoflurane. IMPLICATIONS A subset of serotonin receptors, 5HT2A receptors, may mediate or modulate a minor portion of the immobility produced by inhaled anesthetics.

Several reports suggest that clinically used concentrations of inhaled anesthetics can increase conductance through noninactivating potassium channels and that the resulting hyperpolarization might decrease excitability, thereby leading to the anesthetic state. We speculated that animals deficient in such potassium channels might be resistant to the effects of anesthetics. Thus, in the present study, we measured the minimum alveolar anesthetic concentration (MAC) needed to prevent movement in response to a noxious stimulus in 50% of adult mice lacking functional KCNK5 potassium channel subunits and compared these results with those for heterozygous and wild-type mice. We also measured MAC in weaver mice that had a mutation in the potassium channel Kir3.2 and compared the resulting values with those for wild-type mice. MAC values for desflurane, halothane, and isoflurane for KCNK5-deficient mice and isoflurane MAC values for weaver mice did not differ from MAC values found in control mice. Our results do not support the notion that these potassium channels mediate the capacity of inhaled anesthetics to produce immobility. In addition, we found that the weaver mice did not differ from control mice in their susceptibility to convulsions from the nonimmobilizers flurothyl [di-(2,2,2,-trifluoroethyl)ether] or 2N (1,2-dichlorohexafluorocyclobutane).Mice harboring mutations in either of two different potassium channels have minimum alveolar anesthetic concentration (MAC) values that do not differ from MAC values found in control mice. Such findings do not support the notion that these potassium channels mediate the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation.

Studies suggest that at concentrations surrounding MAC (the minimum alveolar concentration suppressing movement in 50% of subjects in response to noxious stimulation), halothane depresses dorsal horn neurons more than does isoflurane. Similarly, these anesthetics may differ in their effects on various receptors and ion channels that might be anesthetic targets. Both findings suggest that these anesthetics may have effects on movement in response to noxious stimulation that would differ from additivity, possibly producing synergism or even antagonism. We tested this possibility in 20 rats. MAC values for halothane and (separately) for isoflurane were determined in duplicate before and after testing the combination (also in duplicate; six determinations of MAC for each rat). The sum of the isoflurane and halothane MAC fractions for individual rats that produced immobility equaled 1.037 +/- 0.082 and did not differ significantly from a value of 1.00. That is, the combination of halothane and isoflurane produced immobility in response to tail clamp at concentrations consistent with simple additivity of the effects of the anesthetics. These results suggest that the immobility produced by inhaled anesthetics need not result from their capacity to suppress transmission through dorsal horn neurons.Despite differences in their capacities to inhibit spinal dorsal horn cells, isoflurane and halothane are additive in their ability to suppress movement in response to a noxious stimulus.

In animals, the conventional inhaled anesthetic, isoflurane, impairs learning fear to context and fear to tone, doing so at concentrations that produce amnesia in humans. Nonimmobilizers are inhaled compounds that do not produce immobility in response to noxious stimulation, nor do they decrease the requirement for conventional inhaled anesthetics. Like isoflurane, the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N) impairs learning at concentrations less than those predicted from its lipophilicity to produce anesthesia. The capacity of the nonimmobilizer di-(2,2,2,-trifluoroethyl) ether (flurothyl) to affect learning and memory has not been studied. Both nonimmobilizers can cause convulsions. We hypothesized that if isoflurane, 2N, and flurothyl act by the same mechanism to impair learning and memory, their effects should be additive. We found that isoflurane, 2N, and flurothyl (each, alone) impaired learning fear to context and fear to tone in rats, with the nonimmobilizers doing so at concentrations less than those that cause convulsions. (Fear was defined by freezing [volitional immobility] in the presence of the conditioned stimulus [context or tone].) However, the combination of isoflurane and 2N or flurothyl produced an antagonistic rather than an additive effect on learning, a finding in conflict with our hypothesis. And flurothyl was no less potent than 2N (at least no less potent relative to the concentration of each that produced convulsions) in its capacity to impair learning. We conclude that conventional inhaled anesthetics and nonimmobilizers impair learning and memory by different mechanisms. The basis for this impairment remains unknown.Conventional inhaled anesthetics and nonimmobilizers are antagonistic in their effects on learning and memory, and this finding suggests that they impair learning and memory, at least in part, by different mechanisms.

UNLABELLED Many inhaled anesthetics potentiate the effect of glycine on inhibitory strychnine-sensitive glycine receptors in vitro, supporting the view that this receptor could mediate the immobility produced by inhaled anesthetics during noxious stimulation (i.e., would underlie minimum alveolar anesthetic concentration [MAC]). There are quantitative differences between anesthetics in their capacity to potentiate glycine's effect in receptor expression systems: halothane (most potentiation), isoflurane (intermediate), and cyclopropane (minimal). If glycine receptors mediate MAC, then their blockade in the spinal cord should increase the MAC of halothane more than that of isoflurane and isoflurane MAC more than cyclopropane MAC; the increases in MAC should be proportional to the receptor potentiation produced in vitro. Rats with chronically implanted intrathecal catheters were anesthetized with halothane, isoflurane, or cyclopropane. During intrathecal infusion of artificial cerebrospinal fluid, MAC was determined. Then MAC was re-determined during an infusion of 3, 12, 24, or 48 (isoflurane only) micro g/min of strychnine (strychnine blocks glycine receptors) in artificial cerebrospinal fluid. Strychnine infusion increased MAC in proportion to the enhancement of glycine receptors found in vitro. The maximum effect was with an infusion of 12 micro g/min. For the combined results at 12 and 24 micro g/min of strychnine, the increase in MAC correlated with the extent of in vitro potentiation (r(2) = 0.82). These results support the hypothesis that glycine receptors mediate part of the immobilization produced by inhaled anesthetics. IMPLICATIONS In vitro, halothane potentiates glycine's effect on strychnine-sensitive glycine receptors more than isoflurane and isoflurane more than cyclopropane. The present in vivo work indicates that antagonism of the glycine receptor with strychnine increases minimum alveolar anesthetic concentration for halothane more than isoflurane and isoflurane more than cyclopropane. Such results support the notion that glycine receptors may mediate part of the immobility produced by inhaled anesthetics.

UNLABELLED We examined whether N-methyl-D-aspartate (NMDA) receptors influence the immobilizing effect of isoflurane by a spinal or supraspinal action. We antagonized NMDA receptors by intrathecal (IT), intracerebroventricular (ICV), and IV administration of MK 801 (a noncompetitive NMDA antagonist) and measured the decrease in isoflurane minimum alveolar anesthetic concentration (MAC). We also measured MK 801 tissue concentrations in homogenates of upper and lower spinal cord, a slice of cerebral cortex, and the whole brain. IT infusion of MK 801 decreased isoflurane MAC more potently than ICV or IV infusions. The change in MAC correlated with the MK 801 concentration in the lower part of the spinal cord (P < 0.01) but not with concentrations in supraspinal tissue. The maximal effect of IT MK 801 reached a plateau without achieving anesthesia. IV doses 270-fold larger than the largest IT dose also did not produce anesthesia in the absence of isoflurane. These results suggest that the capacity of MK 801 to decrease the MAC of isoflurane results from an effect on the spinal cord but that spinal NMDA receptors provide only partial mediation of the immobility produced by isoflurane. Because neither IT nor IV MK 801 provide complete anesthesia, these findings also call into question the notion that NMDA blockade alone suffices to produce anesthesia as defined by immobility in the face of noxious stimulation. IMPLICATIONS Spinal cord NMDA receptors may mediate a portion of the immobilizing effect of isoflurane. Blockade of NMDA receptors in the cord by MK 801 has a MAC-sparing effect, but MK 801 does not, by itself, produce complete anesthesia.

Background Anesthetic mechanisms of nonhalogenated alkanes cyclopropane and butane are not understood. This study was designed to look at which neurotransmitter receptors are possible targets for these anesthetics. Methods Effects of cyclopropane and butane on eight recombinant receptors expressed in Xenopus oocytes were examined electrophysiologically. To address molecular mechanisms of interaction with glycine and gamma-aminobutyric acid type A (GABA(A)) receptors, cyclopropane was further tested on alpha1(S267C) glycine receptor and alpha2(S270X)beta1 GABA(A) receptors that were mutated to amino acids with larger side chains. Results Cyclopropane (1, 2, and 5 minimum alveolar concentration [MAC]) potentiated glycine responses by 39, 62, and 161%, respectively, and butane (1 MAC) potentiated by 64% with an increase in apparent affinity for glycine, but yielded barely detectable potentiation of GABA(A) receptors. The efficacy of cyclopropane for glycine receptors was less than isoflurane and halothane. The potentiation by cyclopropane was eliminated for the alpha1(S267C) glycine receptor. Mutant GABA(A) receptors in which the corresponding amino acid was substituted with larger amino acids did not produce significant potentiation. Cyclopropane and butane inhibited nicotinic acetylcholine and N-methyl-D-aspartate receptors, potentiated G-protein-coupled inwardly rectifying potassium channels, and did not change 5-hydroxytryptamine(3A) or muscarinic(1) receptor function. Only cyclopropane markedly inhibited alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. Conclusions Glycine, nicotinic acetylcholine, and N-methyl-D-aspartate receptors are sensitive to nonhalogenated alkanes, and the authors propose that glycine and N-methyl-D-aspartate receptors are good candidates for anesthetic immobility. The authors also suggest that the distinct effects on glycine and GABA(A) receptors are not due to the small volumes of these anesthetics.

UNLABELLED Previous reports indicate that cyclosporine increases sleeping time from barbiturate administration, and analgesia from fentanyl administration, suggesting a possible contribution of cyclosporine to anesthesia. Accordingly, we determined whether cyclosporine pretreatment decreases isoflurane MAC (the minimum alveolar concentration of anesthetic required to eliminate movement in response to noxious stimulation in 50% of subjects) in rats, and whether cyclosporine changes the hindpaw withdrawal latency (HPWL) test to thermal nociceptive stimulation. We assigned rats to receive vehicle (control; n = 18), 7.5 mg. kg(-1). d(-1) cyclosporine (n = 10), or 15 mg. kg(-1). d(-1) cyclosporine (n = 20). MAC was determined before and immediately after 3 days of treatment. HPWL was determined after 3 days of treatment in controls (vehicle) and cyclosporine (15 mg. kg(-1). d(-1)) treated animals. Cyclosporine 7.5 and 15 mg. kg(-1). d(-1) increased MAC by 10.4% +/- 7.3% and 23.1% +/- 17.1% (mean +/- SD) (P < 0.05), respectively. The changes in isoflurane MAC correlated significantly with cyclosporine blood concentrations. Cyclosporine did not significantly increase HPWL. Contrary to our hypotheses, cyclosporine increases rather than decreases MAC, but does not confer appreciable analgesia. IMPLICATIONS The immunosuppressive drug, cyclosporine, which is frequently used after organ transplantation, increases the amount of a commonly used anesthetic (isoflurane) necessary to avoid movement to painful stimuli in the rat.

Background Production of retrograde amnesia by anesthetics would indicate that these drugs can disrupt mechanisms that stabilize memory. Such disruption would allow suppression of memory of previous untoward events. The authors examined whether isoflurane provides retrograde amnesia for classic (Pavlovian) fear conditioning. Methods Rats were trained to fear tone by applying three (three-trial) or one (one-trial) tone-shock pairs while breathing various constant concentrations of isoflurane. Immediately after training, isoflurane administration was either discontinued, maintained unchanged, or rapidly increased to 1.0 minimum alveolar concentration for 1 h longer. Groups of rats were similarly trained to fear context while breathing isoflurane by applying shocks (without tones) in a distinctive environment. The next day, memory for the conditioned stimuli was determined by presenting the tone or context (without shock) and measuring the proportion of time each rat froze (appeared immobile). For each conditioning procedure, the effects of the three posttraining isoflurane treatments were compared. Results Rapid increases in posttraining isoflurane administration did not suppress conditioned fear for any of the training procedures. In contrast, isoflurane administration during conditioning dose-dependently suppressed conditioning (P < 0.05). Training to tone was more resistant to the effects of isoflurane than training to context (P < 0.05), and the three-trial learning procedure was more was more resistant than the one-trial procedure (P < 0.05). Conclusions Isoflurane provided intense dose-dependent anterograde but not retrograde amnesia for classic fear conditioning. Isoflurane appears to disrupt memory processes that occur at or within a few minutes of the conditioning procedure.

UNLABELLED: The in vivo potencies of anesthetics correlate with their capacity to suppress the reaction of luciferin with luciferase. In addition, luciferin has structural resemblances to etomidate. These observations raise the issues of whether luciferin, itself, might affect anesthetic requirement, and whether luciferase resembles the site of anesthetic action. Because the polar luciferin is unlikely to cross the blood-brain barrier (we found that the olive oil/water partition coefficient was 100 +/- 36 x 10(-7)), we studied these issues in rats by measuring the effect of infusion of luciferin in artificial cerebrospinal fluid into the lumbar subarachnoidal space and into the cerebral intraventricular space on the MAC (the minimum alveolar anesthetic concentration required to eliminate movement in response to a noxious stimulus in 50% of tested subjects) of isoflurane. MAC in rats given lumbar intrathecal doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate, did not differ significantly from MAC in rats receiving only artificial cerebrospinal fluid into the lumbar intrathecal space. MAC slightly decreased when doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate were infused intraventricularly (P < 0.05). In contrast to the absent or minimal effects of luciferin, intrathecal or intraventricular infusion of etomidate at similar or smaller doses significantly decreased isoflurane MAC. Luciferin did not affect +-aminobutyric acid type A or acetylcholine receptors expressed in Xenopus oocytes. These results suggest that luciferin has minimal or no anesthetic effects. It also suggests that luciferin/luciferase may not provide a good surrogate for the site at which anesthetics act, if this site is on the surface of neuronal cells. IMPLICATIONS: In proportion to their potencies, anesthetics inhibit luciferin's action on luciferase, and luciferin structurally resembles the anesthetic etomidate. However, in contrast to etomidate, luciferin given intrathecally or into the third cerebral ventricle does not have anesthetic actions, and it does not affect +-aminobutyric acid or acetylcholine receptors in vitro. Luciferase may not provide a good surrogate for the site at which anesthetics act.

Two defining effects of inhaled anesthetics (immobility in the face of noxious stimulation, and absence of memory) correlate with the end-tidal concentrations of the anesthetics. Such defining effects are characterized as MAC (the concentration producing immobility in 50% of patients subjected to a noxious stimulus) and MAC-Awake (the concentration suppressing appropriate response to command in 50% of patients; memory is usually lost at MAC-Awake). If the concentrations are monitored and corrected for the effects of age and temperature, the concentrations may be displayed as multiples of MAC for a standard age, usually 40 yr. This article provides an algorithm that might be used to produce such a display, including provision of an estimate of the effect of nitrous oxide.Two defining effects of inhaled anesthetics (immobility in the face of noxious stimulation, and absence of memory) correlate with the end-tidal concentrations of the anesthetics. Thus, these defining effects may be monitored and the results displayed if the concentrations are known and corrected for the effects of age and temperature.