Inhaled anesthetic responses of recombinant receptors and knockin mice harboring α2(S270H/L277A) GABA(A) receptor subunits that are resistant to isoflurane.

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.

Blockade of AMPA receptors and volatile anesthetics: reduced anesthetic requirements in GluR2 null mutant mice for loss of the righting reflex and antinociception but not minimum alveolar concentration.

BACKGROUND: The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor mediates fast excitatory neurotransmission in the central nervous system. Many general anesthetics inhibit AMPA receptors in vitro; however, it is not certain if this inhibition contributes to the behavioral properties of these drugs. AMPA receptors lacking the GluR2 subunit are resistant to blockade by barbiturates in vitro. Paradoxically, GluR2 null mutant (-/-) mice are more sensitive to barbiturate-induced loss of the righting reflex (LORR) compared with wild-type (+/+) littermates. To determine if interactions between anesthetics and AMPA receptors account for the increased sensitivity of (-/-) mice, the effects of volatile anesthetics that do not directly inhibit AMPA receptors were examined. METHODS: Isoflurane, halothane, desflurane, or sevoflurane were administered to (-/-) and (+/+) littermate controls. Anesthetic requirements for LORR, movement to tail clamp (minimum alveolar concentration [MAC]), and hind-paw withdrawal latency (HPWL) were determined. Electrophysiologic methods examined the inhibition of AMPA receptors by isoflurane and halothane. RESULTS: Anesthetic requirements for LORR and HPWL were decreased, whereas MAC values were unchanged in (-/-) mice. Isoflurane and halothane caused minimal inhibition of AMPA receptors at clinically relevant concentrations. CONCLUSIONS: Direct blockade of AMPA receptors did not account for the increased sensitivity to volatile anesthetics in GluR2 null mutant mice for HPWL or LORR. Thus, the deficiency of GluR2-containing AMPA receptors increases the sensitivity of neuronal circuitry mediating these end points, but not MAC. GluR2-containing receptors do not contribute appreciably to MAC in this mouse model. These results illustrate the difficulties in attributing behavioral responses to drug-receptor interactions in genetically engineered animals.

The concentration of isoflurane required to suppress learning depends on the type of learning.

BACKGROUND: Recent reports suggest that one type of learning, fear conditioning to context, requires more neural processing than a related type, fear conditioning to tone. To determine whether these types of learning were differentially affected by anesthesia, the authors applied isoflurane during the training phases of fear conditioning paradigms for freezing to context and freezing to tone. METHODS: The authors trained seven groups of eight rats to fear tone by administering a tone (conditioned stimulus) while breathing various concentrations of isoflurane from 0.00 to 0.75 minimum alveolar concentration (MAC; one concentration per group) separated by 0.12-MAC steps. On the succeeding day, and in the absence of isoflurane, the authors presented the tone (without shock) in a different context (different cage shape and odor) and measured the time each rat froze (became immobile). Six other groups of eight rats were trained to fear context by applying the shock in the absence of a tone but in the presence of environmental cues such as cage shape, texture, and odor. Fear to context was determined the succeeding day by returning the rat to the training cage (without shock) and measuring duration of freezing. Control groups (16 per group) received 0.75 MAC isoflurane but no foot shocks. Group scores were compared using analysis of variance, and the ED50 values for quantal responses of individual rats were calculated using logistic regression. RESULTS: Conditioning to context occurred at 0.00 and 0.13 MAC (P < 0.05 compared with unshocked control) but not 0.25 MAC; the ED50 was 0.25 +/- 0.03 MAC (mean +/- SEM). In contrast, conditioning to tone occurred at 0.48 MAC (P < 0.05) but not 0.62 MAC; the ED50 was 0.47 +/- 0.02 MAC (P < 0.01 for the difference between ED50 values). CONCLUSIONS: Suppression of fear conditioning to tone required approximately twice the isoflurane concentration that suppressed fear conditioning to context. Thus, the concentration of anesthetic required to suppress learning may depend on the neural substrates of learning. Our results suggest that isoflurane concentrations greater than 0.5 MAC may be needed to suppress both forms of fear conditioning.

Neither GABA(A) nor strychnine-sensitive glycine receptors are the sole mediators of MAC for isoflurane.

UNLABELLED: Inhaled anesthetics produce immobility (a cardinal aspect of general anesthesia) by an action on the spinal cord, possibly by potentiating the responses of gamma-amino-n-butyric acid (GABA(A)) and glycine receptors to GABA and glycine. In this study, we antagonized GABA(A) and glycine responses by intrathecal administration of picrotoxin (a noncompetitive GABA(A) antagonist), strychnine (a competitive glycine antagonist), or combinations of these drugs. We measured the capacity of antagonist infusion to increase isoflurane MAC (the minimum alveolar concentration of anesthetic that prevents movement in response to noxious stimuli in 50% of subjects). We found that these potent GABA(A) and glycine receptor antagonists had a ceiling effect, either alone or in combination increasing the MAC of isoflurane by at most 47%. IMPLICATIONS: gamma-amino-n-butyric acid and glycine receptors may in part be responsible for the immobilizing action of isoflurane. They are not, however, the only receptors that contribute to isoflurane-induced immobility (i.e., that determine the MAC of isoflurane).

The nonimmobilizer 1,2-dichlorohexafluorocyclobutane does not affect thermoregulation in the rat.

Inhaled and other anesthetics profoundly affect the central nervous system, causing amnesia, immobility in the face of noxious stimulation, and depression of thermoregulation. Nonimmobilizers, inhaled compounds whose lipophilicity suggests that they should be anesthetics, do not produce immobility, but they do cause amnesia. Their effects on thermoregulation were the subject of the present study. We gave eight rats isoflurane on one occasion and the nonimmobilizer 2N (1,2-dichlorolhexafluorocyclobutane) on another. We measured the effect of various concentrations of each compound on thermoregulation provoked by body cooling. The specific outcome was increased metabolism, as reflected in increased output of carbon dioxide. Isoflurane decreased the temperature threshold for such increases and the maximum response intensity, doing so in a concentration-dependent manner, whereas 2N had a minimal or no effect at any concentration up to 0.9 minimum alveolar concentration (estimated from its lipophilicity). Thus, 2N may be a useful tool for studies of the mechanisms mediating the thermoregulatory depression produced by anesthetics: 2N should not affect such a mechanism.

Naturally occurring variability in anesthetic potency among inbred mouse strains.

UNLABELLED: We measured the naturally occurring variability in anesthetic potency, defined by the minimum alveolar anesthetic concentrations (MACs) of inhaled anesthetics required to produce immobility in response to noxious stimuli, in seven widely used laboratory mouse strains. To these data, we added similar data for eight other mouse strains. The average MAC values for each anesthetic for the 15 strains were normally distributed, with a coefficient of variation (ratio of SD to mean) of 0.1. The range of MAC values was 39% for desflurane, 44% for isoflurane, and 55% for halothane. MAC values were highly reliable, with approximately 1% of the variance in MAC measurements for the strains being explained by measurement error. One hundred forty-six statistically significant differences among the 15 strains were found for the three inhaled anesthetics (isoflurane, desflurane, and halothane). Our results suggest that multiple genes underlie the observed variability in anesthetic potency. IMPLICATIONS: Laboratory mouse strains differ significantly in susceptibility to anesthetics. These phenotypic differences may be exploited to help determine the genetic basis of anesthetic-induced immobility.

Inhaled anesthetics have hyperalgesic effects at 0.1 minimum alveolar anesthetic concentration.

UNLABELLED: We investigated the hyperalgesic (antianalgesic) effect of the inhaled anesthetics isoflurane, halothane, nitrous oxide, and diethyl ether, or the nonimmobilizer 1, 2-dichlorohexafluorocyclobutane at subanesthetic partial pressures (or, for the nonimmobilizer, subanesthetic partial pressures predicted from lipid solubility) in rats. Hyperalgesia was assessed as a decrease in the time to withdrawal of a rat hind paw exposed to heat. All four anesthetics, including nitrous oxide and diethyl ether, produced hyperalgesia at low partial pressures, with a maximal effect at 0.1 minimum alveolar anesthetic concentration (MAC) required to prevent response to movement in 50% of animals, and analgesia (an increased time to withdrawal of the hind paw) at 0. 4 to 0.8 MAC. The nonimmobilizer had neither analgesic nor hyperalgesia effects. We propose that inhaled anesthetics with a higher MAC-Awake (the MAC-fraction that suppresses appropriate responsiveness to command), such as nitrous oxide and diethyl ether, can be used as analgesics because patients are conscious at higher anesthetic partial pressures, including those which have analgesic effects, whereas anesthetics with a lower MAC-Awake do not produce analgesic effects at concentrations that permit consciousness. IMPLICATIONS: The inhaled anesthetics isoflurane, halothane, nitrous oxide, and diethyl ether produce antianalgesia at subanesthetic concentrations, with a maximal effect at approximately one-tenth the concentration required for anesthesia. This effect may enhance perception of pain when such small concentrations are reached during recovery from anesthesia.

Mouse strain modestly influences minimum alveolar anesthetic concentration and convulsivity of inhaled compounds.

UNLABELLED: In this study, we measured the minimum alveolar anesthetic concentration (MAC) in several mouse strains, including strains used in the construction of genetically engineered mice. This is important because defined genetic modifications are used increasingly to test mechanisms of inhaled anesthetic action, and background variability in MAC can potentially influence the interpretation of these studies. We investigated the effect of strain on MAC for desflurane, isoflurane, halothane, ethanol, the experimental anesthetic 1-chloro-1,2,2-trifluorocyclobutane, and convulsive 50% effective dose (the dose required to produce convulsions in 50% of animals) of the nonimmobilizer 1,2-dichlorohexafluorocyclobutane. These drugs were studied in eight inbred strains, including both laboratory and wild mouse strains (129/J, 129/SvJ, 129/Ola Hsd, C57BL/6NHsd, C57BL/6J, DBA/2J, Spret/Ei, and Cast/Ei), one hybrid strain (B6129F2/J, derived from the C57BL/6J and 129/J strains), and one outbred strain (CD-1). To test our ability to detect effects in a genetically modified mouse, we compared these data with those for a mouse lacking the gamma (neuronal) isoform of the protein kinase C gene (PKCgamma). We also assessed whether amputating the tail tip of mice (a standard method of obtaining tissue for genetic analysis) increased MAC (e.g., by sensitization of the spinal cord). MAC and convulsant 50% effective dose values differed modestly among strains, with a range of 17% to 39% from the lowest to highest values for MAC using conventional anesthetics, and up to 48% using the experimental anesthetic 1-chloro-1,2,2-trifluorocyclobutane. Convulsivity to the nonimmobilizer varied by 47%. Amputating the tail tip did not affect MAC. PKCgamma knockout mice had significantly higher MAC values than control animals for isoflurane, but not for halothane or desflurane, which implies that protein phosphorylation by PKCgamma can alter sensitivity to isoflurane. IMPLICATIONS: Anesthetic potency differs by modest amounts among inbred, outbred, wild, and laboratory mouse strains. Absence of the neural form of protein kinase C increases minimum alveolar anesthetic concentration for isoflurane, indicating that protein phosphorylation by the gamma-isoform of protein kinase C (PKCgamma) can influence the potency of this anesthetic.

Hypothesis: volatile anesthetics produce immobility by acting on two sites approximately five carbon atoms apart.

UNLABELLED: All series of volatile and gaseous compounds contain members that can produce anesthesia, as defined by the minimum alveolar anesthetic concentration (MAC) required to produce immobility in response to a noxious stimulus. For unhalogenated n-alkanes, cycloalkanes, aromatic compounds, and n-alkanols, potency (1 MAC) increases by two-to threefold with each carbon addition in the series (e.g., ethanol is twice as potent as methanol). Total fluorination (perfluorination) of n-alkanes essentially eliminates anesthetic potency: only CF4 is anesthetic (MAC = 66.5 atm), which indicates that fluorine atoms do not directly influence sites of anesthetic action. Fluorine may enhance the anesthetic action of other moieties, such as the hydrogen atom in CHF3 (MAC = 1.60 atm), but, consistent with the notion that the fluorine atoms do not directly influence sites of anesthetic action, adding -(CF2)n moieties does not further increase potency (e.g., CHF2-CF3 MAC = 1.51 atm). Similarly, adding -(CF2)n moieties to perfluorinated alkanols (CH2OH-[CF2]nF) does not increase potency. However, adding a second terminal hydrogen atom (e.g., CHF2-CHF2 or CH2OH-CHF2) produces series in which the addition of each -CF2- "spacer" in the middle of the molecule increases potency two- to threefold, as in each unhalogenated series. This parallel stops at four or five carbon atom chain lengths. Further increases in chain length (i.e., to CHF2[CF2]4CHF2 or CHF2[CF2]5CH2OH) decrease or abolish potency (i.e., a discontinuity arises). This leads to our hypothesis that the anesthetic moieties (-CHF2 and -CH2OH) interact with two distinct, spatially separate, sites. Both sites must be influenced concurrently to produce a maximal anesthetic (immobility) effect. We propose that the maximal potency (i.e., for CHF2[CF2]2CHF2 and CHF2[CF2]3CH2OH) results when the spacing between the anesthetic moieties most closely matches the distance between the two sites of action. This reasoning suggests that a distance equivalent to a four or five carbon atom chain, approximately 5 A, separates the two sites. IMPLICATIONS: Volatile anesthetics may produce immobility by a concurrent action on two sites five carbon atom lengths apart.

Ethanol concentrations approaching minimum alveolar anesthetic concentration are required to suppress learning in a fear-potentiated startle paradigm in rats.

UNLABELLED: We previously demonstrated that desflurane and two nonimmobilizers dose-dependently decrease learning and memory in rats. This suggests that although they do not suppress movement in response to noxious stimuli, nonimmobilizers act like inhaled anesthetics in their effects on learning and memory. Like most conventional anesthetics, nonimmobilizers have a greater affinity for lipid than for aqueous phases. In the present study, we examined the effect of ethanol on learning and memory to test the hypothesis that a large part of the capacity of anesthetics to affect learning depends on an action on a lipid (nonpolar) phase. Unlike volatile anesthetics and nonimmobilizers, ethanol has a greater affinity for water than for lipids. Thus, if our hypothesis is correct, ethanol should be relatively less potent in its suppression of memory. Rats receiving various doses of ethanol were conditioned to fear a light followed by a footshock. Fear conditioning to the light was subsequently assessed by measurement of potentiation of the acoustic startle reflex in the presence, compared with the absence, of light. Ethanol up to 0.54 minimum alveolar anesthetic concentration (MAC) did not abolish fear, but 0.82 MAC ethanol did abolish learning. Expressed as a fraction of MAC or predicted MAC, ethanol is less potent than desflurane or the nonimmobilizer 1,2-dichlorohexafluorocyclobutane in suppressing learning. This finding is consistent with the hypothesis that the capacity of anesthetics and nonimmobilizers to impair learning and memory depends mostly on an action at a nonpolar site. IMPLICATIONS: Abolition of learning and memory is an important property of inhaled anesthetics. This effect primarily results from an action at a lipid (nonpolar) site, rather than a polar site or a water-lipid interface.

Desflurane and the nonimmobilizer 1,2-dichlorohexafluorocyclobutane suppress learning by a mechanism independent of the level of unconditioned stimulation.

UNLABELLED: We previously demonstrated that anesthetics and non-immobilizers suppress learning and memory in rats. In the training portion of the test, rats received a light plus a footshock and learned to associate the two, as evidenced by subsequent potentiation of the response (jumping) to light plus a noise (fear-potentiated startle). However, anesthetics and nonimmobilizers also decreased the response of animals receiving footshocks during training, which suggests that the reduction in fear-potentiated startle might reflect analgesia, rather than an impairment of learning and memory. Furthermore, although we previously demonstrated that the nonimmobilizer 2,3-dichlorohexafluorocyclobutane (2N) could completely abolish learning, we did not demonstrate the minimal dose required. In the present study, we eliminated analgesia as a confounding factor by training rats breathing desflurane and 2N with footshock intensities that produced responses at least equal to those produced in control animals. Both desflurane and 2N suppressed learning at 0.2 times the minimum alveolar anesthetic concentration (MAC) or the MAC predicted from lipid solubility, despite the increased footshock intensity. This partial pressure of desflurane equals that previously shown to suppress learning at lower footshock intensities. We conclude that suppression of learning and memory by desflurane and 2N does not result from decreased sensitivity to the unconditioned stimulus (the footshock) and that the potency of 2N is consistent with its lipophilicity. IMPLICATIONS: General anesthesia eliminates recall of intraoperative events, including pain. Using an animal model, we refuted the hypothesis that lack of recall results from the analgesia (i.e., the reduced response to painful stimuli produced by inhaled drugs) rather than from a direct effect on learning.

Nausea and vomiting following thyroid and parathyroid surgery.

STUDY OBJECTIVES: To determine the incidence of postoperative nausea and vomiting (PONV) following thyroid and parathyroid surgery. To determine whether PONV is reduced when propofol is used for maintenance of anesthesia as compared to isoflurane and to evaluate the costs and resource consumption associated with these two anesthetic regimens. DESIGN: Randomized, prospective study. SETTING: University-affiliated hospital--a referral center for endocrinologic surgery. PATIENTS: 118 ASA physical status I and II patients, aged 18 years and older, undergoing elective thyroid or parathyroid surgery. INTERVENTIONS: Patients received either isoflurane (0.5 to 1.3% end-tidal) or propofol (50 to 200 micrograms/kg/min) for maintenance of anesthesia. All patients received propofol for induction of anesthesia, succinylcholine or vecuronium, nitrous oxide, and fentanyl. Prophylactic antiemetics were not administered. Postoperative pain was treated with ketorolac, fentanyl, or acetaminophen. MEASUREMENTS AND MAIN RESULTS: Signs and symptoms of nausea and vomiting were graded on a four point scale as 1 = no nausea; 2 = mild nausea; 3 = severe nausea; 4 = retching and/or vomiting. Grades 3 and 4 were grouped together as PONV. The combined incidence of PONV was 54% over the 24-hour postoperative evaluation period. PONV was significantly more common in patients receiving isoflurane than propofol for maintenance of anesthesia (64% vs. 44%). In women (n = 87), the incidence of PONV was significantly greater in those patients who received isoflurane than those who received propofol for maintenance (71% vs. 42%). However, in men (n = 31), there was no significant difference in PONV between anesthetic regimens (47% with isoflurane vs. 50% with propofol). There were no differences in the duration of stay in the postanesthesia care unit, time to discharge from the hospital, or local wound complications (hematomas) between groups. The use of propofol for maintenance of anesthesia was associated with an additional cost, relative to the isoflurane group, of $54.26 per patient. CONCLUSION: Patients undergoing thyroid or parathyroid surgery are at high risk for the development of PONV. Propofol for maintenance of anesthesia, although more expensive than isoflurane, reduces the rate of PONV in women.

Natural Occurrence of Indole-3-acetylaspartate and Indole-3-acetylglutamate in Cucumber Shoot Tissue.

Indole-3-acetylaspartate and indole-3-acetylglutamate were isolated from 1-week-old, green cucumber shoots that had not been pretreated with auxin. Using isotope dilution techniques, we found 129 micrograms (0.42 micromoles) of indoleacetylglutamate and 33 micrograms (0.11 micromoles) of indoleacetylaspartate per kilogram of fresh tissue.