Yilei Xing

Gabaa Receptor Blockade Antagonizes the Immobilizing Action of Propofol but Not Ketamine or Isoflurane in a Dose-related Manner

The enhancing action of propofol on &ggr;-amino-n-butyric acid subtype A (GABAA) receptors purportedly underlies its anesthetic effects. However, a recent study found that a GABAA antagonist did not alter the capacity of propofol to depress the righting reflex. We examined whether the noncompetitive GABAA antagonist picrotoxin and the competitive GABAA antagonist gabazine affected a different anesthetic response, immobility in response to a noxious stimulus (a tail clamp in rats), produced by propofol. This effect was compared with that seen with ketamine and isoflurane. Picrotoxin increased the 50% effective dose (ED50) for propofol by approximately 379%; gabazine increased it by 362%, and both antagonists acted in a dose-related manner with no apparent ceiling effect (i.e., no limit). Picrotoxin maximally increased the ED50 for ketamine by approximately 40%–50%, whereas gabazine increased it by 50%–60%. The isoflurane minimum alveolar anesthetic concentration increased by approximately 60% with the picrotoxin and 70% with the gabazine infusion. The ED50 for propofol was also antagonized by strychnine, a non-GABAergic glycine receptor antagonist and convulsant, to determine whether excitation of the central nervous system by a non-GABAergic mechanism could account for the increases in propofol ED50 observed. Because strychnine only increased the immobilizing ED50 of propofol by approximately 50%, GABAA receptor antagonism accounted for the results seen with picrotoxin and gabazine. We conclude that GABAA antagonism can influence the ED50 for immobility of propofol and the non-GABAergic anesthetic ketamine, although to a different degree, reflecting physiologic antagonism for ketamine (i.e., an indirect effect via a modulatory effect on the neural circuitry underlying immobility) versus physiologic and pharmacologic antagonism for propofol (i.e., a direct effect by antagonism of propofol’s mechanism of action). This study also suggests that the immobilizing action of isoflurane probably does not involve the GABAA receptor because antagonism of GABAA receptors for animals anesthetized with isoflurane produces results quantitatively and qualitatively similar to ketamine and markedly different from propofol.

Effect of isoflurane and other potent inhaled anesthetics on minimum alveolar concentration, learning, and the righting reflex in mice engineered to express α1 γ-aminobutyric acid type A receptors unresponsive to isoflurane

Background:Enhancement of the function of γ-aminobutyric acid type A receptors containing the α1 subunit may underlie a portion of inhaled anesthetic action. To test this, the authors created gene knock-in mice harboring mutations that render the receptors insensitive to isoflurane while preserving sensitivity to halothane. Methods:The authors recorded miniature inhibitory synaptic currents in hippocampal neurons from hippocampal slices from knock-in and wild-type mice. They also determined the minimum alveolar concentration (MAC), and the concentration at which 50% of animals lost their righting reflexes and which suppressed pavlovian fear conditioning to tone and context in both genotypes. Results:Miniature inhibitory postsynaptic currents decayed more rapidly in interneurons and CA1 pyramidal cells from the knock-in mice compared with wild-type animals. Isoflurane (0.5–1 MAC) prolonged the decay phase of miniature inhibitory postsynaptic currents in neurons of the wild-type mice, but this effect was significantly reduced in neurons from knock-in mice. Halothane (1 MAC) slowed the decay of miniature inhibitory postsynaptic current in both genotypes. The homozygous knock-in mice were more resistant than wild-type controls to loss of righting reflexes induced by isoflurane and enflurane, but not to halothane. The MAC for isoflurane, desflurane, and halothane did not differ between knock-in and wild-type mice. The knock-in mice and wild-type mice did not differ in their sensitivity to isoflurane for fear conditioning. Conclusions:γ-Aminobutyric acid type A receptors containing the α1 subunit participate in the inhibition of the righting reflexes by isoflurane and enflurane. They are not, however, involved in the amnestic effect of isoflurane or immobilizing actions of inhaled agents.

Spinal N-methyl-d-aspartate receptors may contribute to the immobilizing action of isoflurane.

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.

Mice with a Melanocortin 1 Receptor Mutation Have a Slightly Greater Minimum Alveolar Concentration than Control Mice

ANESTHESIA folklore includes a perception that patients with red hair have a greater MAC (the minimum alveolar concentration of anesthetic that prevents movement in response to noxious stimuli in 50% of subjects). In support of this perception, Liem et al. found that a greater concentration of the inhaled anesthetic desflurane was required to suppress movement in response to intense electrical stimulation in red haired humans. Such a finding has obvious clinical implications. In addition, a determination of the underlying cause might provide some insight into the mechanisms by which inhaled anesthetics act. Loss of function mutations in the melanocortin 1 receptor (MC1R) gene account for the majority of cases of red hair in humans. Mice with a melanocortin 1 receptor mutation (MC1R) resulting in a nonfunctional receptor have a yellow coat. These observations suggested the hypothesis that MC1R mice have greater MAC values than control mice. Accordingly, we determined desflurane, isoflurane, halothane, and sevoflurane MAC values for both MC1R and control mice.

Isoflurane antagonizes the capacity of flurothyl or 1,2-dichlorohexafluorocyclobutane to impair fear conditioning to context and tone.

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.

Do N-methyl-D-aspartate receptors mediate the capacity of inhaled anesthetics to suppress the temporal summation that contributes to minimum alveolar concentration?

Antagonism of N-methyl-d-aspartate (NMDA) receptors markedly decreases the minimum alveolar concentration (MAC) of inhaled anesthetics. To assess the importance of suppression of the temporal summation NMDA receptor component of MAC, we stimulated the tail of rats with trains of electrical pulses of varying interstimulus intervals (ISIs) and determined the inhaled anesthetic concentrations (crossover concentrations) that suppressed movement at different ISIs. The slopes of crossover concentrations versus ISIs provided a measure of temporal summation for each anesthetic. We studied five anesthetics that differ widely in their in vitro capacity to block NMDA receptors. To block NMDA receptor transmission and reveal the NMDA receptor component, the NMDA receptor antagonist, MK801, was separately added during each anesthetic. Halothane, isoflurane, and hexafluorobenzene did not appreciably suppress the NMDA receptor components of temporal summation, which contributed to 21% to 29% of MAC (P < 0.05 for each). Xenon and o-difluorobenzene suppressed these components to 8% to 0%, respectively, of MAC (neither significant), consistent with their greater NMDA receptor blocking action in vitro. NMDA receptor blockade may contribute to the MAC produced by inhaled anesthetics that potently inhibit NMDA receptors in vitro but not those that have a limited in vitro effect.

Mouse chromosome 7 harbors a quantitative trait locus for isoflurane minimum alveolar concentration.

BACKGROUND: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. METHODS: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. RESULTS AND CONCLUSIONS:Both methods of genetic analysis identified a QTL for isoflurane MAC that was located on the proximal part of mouse chromosome 7.

Administration of Epinephrine Does Not Increase Learning of Fear to Tone in Rats Anesthetized with Isoflurane or Desflurane

Previous reports suggest that the administration of epinephrine increases learning during deep barbiturate-chloral hydrate anesthesia in rats but not during anesthesia with 0.4% isoflurane in rabbits. We revisited this issue, using fear conditioning to a tone in rats as our experimental model for learning and memory and isoflurane and desflurane as our anesthetics. Expressed as a fraction of the minimum alveolar anesthetic concentration (MAC) preventing movement in 50% of rats, the amnestic 50% effective dose (ED50) for fear to tone in control rats inhaling isoflurane and injected with saline intraperitoneally (i.p.) was 0.32 ± 0.03 MAC (mean ± se) compared with 0.37 ± 0.06 MAC in rats injected with 0.01 mg/kg of epinephrine i.p. and 0.38 ± 0.03 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. For desflurane, the amnestic ED50 were 0.32 ± 0.05 MAC in control rats receiving a saline injection i.p. versus 0.36 ± 0.04 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. We conclude that exogenous epinephrine does not decrease amnesia produced by inhaled isoflurane or desflurane, as assessed by fear conditioning to a tone in rats.

Acetylcholine receptors and thresholds for convulsions from flurothyl and 1,2-dichlorohexafluorocyclobutane.

There are acetylcholine receptors throughout the central nervous system, and they may mediate some forms and aspects of convulsive activity. Most high-affinity binding sites on nicotinic acetylcholine receptors for nicotine, cytisine, and epibatidine in the brain contain the &bgr;2 subunit of the receptor. Transitional inhaled compounds (compounds less potent than predicted from their lipophilicity and the Meyer-Overton hypothesis) and nonimmobilizers (compounds that do not produce immobility despite a lipophilicity that suggests anesthetic qualities as predicted from the Meyer-Overton hypothesis) can produce convulsions. The nonimmobilizer flurothyl [di-(2,2,2,-trifluoroethyl)ether] blocks the action of &ggr;-aminobutyric acid on &ggr;-aminobutyric acidA receptors, whereas the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N, also called F6) does not. 2N can block the action of acetylcholine on nicotinic acetylcholine receptors. We examined the relative capacities of these compounds to cause convulsions in mice having and lacking the &bgr;2 subunit of the acetylcholine receptor. The partial pressure causing convulsions in half the mice (the 50% effective concentration [EC50]) was the same as in control mice. For the knockout mice, the EC50 for flurothyl was 0.00170 ± 0.00030 atm (mean ± sd), and for 2N, it was 0.0345 ± 0.0041 atm. For the control mice, the respective values were 0.00172 ± 0.00057 atm and 0.0341 ± 0.0048 atm. The ratio of the 2N to flurothyl EC50 values was 20.8 ± 3.5 for the knockout mice and 21.7 ± 7.0 for the control mice. These results do not support the notion that acetylcholine receptors are important mediators of the capacity of 2N or flurothyl to cause convulsions. However, we also found that both nonimmobilizers inhibit rat &agr;4&bgr;2 neuronal nicotinic acetylcholine receptors at EC50 partial pressures (0.00091 atm and 0.062 atm for flurothyl and 2N, respectively) that approximate those that produce convulsions (0.0015 atm and 0.04 atm).