Search

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.

Inhaled anesthetics are believed to produce anesthesia by their actions on ion channels. Because inhaled anesthetics robustly enhance GABA A receptor (GABA(A)-R) responses to GABA, these receptors are considered prime targets of anesthetic action. However, the importance of GABA(A)-Rs and individual GABA(A)-R subunits to specific anesthetic-induced behavioral effects in the intact animal is unknown. We hypothesized that inhaled anesthetics produce amnesia, as assessed by loss of fear conditioning, by acting on the forebrain GABA(A)-Rs that harbor the alpha1 subunit. To test this, we used global knockout mice that completely lack the alpha1 subunit and forebrain-specific, conditional knockout mice that lack the alpha1 subunit only in the hippocampus, cortex, and amygdala. Both knockout mice were 75 to 145% less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that alpha1-containing GABA(A)-Rs in the hippocampus, amygdala, and/or cortex influence the amnestic effects of inhaled anesthetics and may be an important molecular target of the drug isoflurane.

Inhalable solvents possess significant abuse liability and produce many of the neurobehavioral effects typically associated with central nervous system-depressant agents, including motor incoordination, anxiolysis, and the elicitation of signs of physical dependence on withdrawal. We tested the hypothesis that the commonly abused solvents toluene, 1,1,1-trichloroethane (TCE), and trichloroethylene (TCY) affect ligand-gated ion channel activity, as do other classes of central nervous system-depressive agents. TCE and toluene, like ethanol, reversibly enhanced gamma-aminobutyric acid (GABA)(A) receptor-mediated synaptic currents in rat hippocampal slices. All three inhalants significantly and reversibly enhanced neurotransmitter-activated currents at alpha1beta1 GABA(A) and alpha1 glycine receptors expressed in Xenopus oocytes. We previously identified specific amino acids of glycine and GABA(A) receptor subunits mediating alcohol and volatile anesthetic enhancement of receptor function. Toluene, TCE, and TCY were tested on several glycine receptor mutants, some of which were insensitive to ethanol and/or enflurane. Toluene and TCY enhancement of glycine receptor function was seen in all these mutants. However, the potentiating effects of TCE were abolished in three mutants and enhanced in two, a pattern more akin to that seen with enflurane than ethanol. These data suggest that inhaled drugs of abuse affect ligand-gated ion channels, and that the molecular sites of action of these compounds may overlap with those of ethanol and the volatile anesthetics.

We assessed the involvement of specific glutamate receptors in the action of anesthetics. In addition to the clinical anesthetics enflurane, isoflurane and halothane, we tested novel halogenated compounds, which are anesthetic or nonanesthetic in vivo, on glutamate receptor (GluR) subunits. these volatile compounds as well as pentobarbital and phenobarbital were tested on kainate-induced currents in Xenopus oocytes expressing GluR1, GluR3, GluR2+3 or GluR6 subunits. The anesthetic 1-chloro-1,2,2-triflurocyclobutane (F3) weakly inhibited kainate responses in oocytes expressing GluR3 receptors but not oocytes expressing GluR1 or GluR2+3 receptors. Surprisingly, F3 potentiated kainate responses in oocytes expressing the kainate-selective receptor GluR6. The nonanesthetics 2,3-chlorooctafluorobutane (F8) and 1,2-dichlorohexafluorocyclobutane (F6) did not affect GluR3 or GluR6 kainate responses. Isoflurane weakly inhibited although enflurane and halothane modestly inhibited kainate responses in oocytes expressing GluR1, GluR3 or GluR2+3 receptors. As with F3, isoflurane, enflurane and halothane potentiated kainate-induced currents of GluR6 receptors. The respective inhibitory and potentiating effects of halothane on GluR3 and GluR6 receptors were enhanced by increasing duration of halothane exposure. In contrast to the opposing action of volatile anesthetics, pentobarbital and phenobarbital inhibited GluR3 and GluR6 kainate responses and had a much greater effect on GluR6 receptors. These results provide novel evidence that anesthetics produce selective actions on glutamate receptors, suggesting that subunit composition may determine the role of glutamate receptors in anesthesia.

The Meyer-Overton hypothesis, predicting that the potency of an anesthetic correlates with its affinity for lipid, is a cornerstone of modern anesthetic theory. Several halogenated compounds were recently found to deviate from this prediction, whereas others did not. We tested the abilities of enflurane and five of these compounds to potentiate gamma-aminobutyric acid (GABA)A receptor responses in Xenopus oocytes expressing alpha 1 beta 2 or alpha 1 beta 2 gamma 2S GABAA receptors. Enflurane and the anesthetic 1-chloro-1,2,2-trifluorocyclobutane (F3) strongly potentiated chloride currents produced by 5 microM GABA with both alpha 1 beta 2 and alpha 1 beta 2 gamma 2S receptors. This potentiation decreased as the GABA concentration was raised. The transitional compound (less potent than predicted by its lipid solubility) 2-bromoheptafluoropropane produced modest enhancement, whereas three nonanesthetics (neither causing anesthesia in vivo nor decreasing the requirement for known anesthetics), 1,2-dichlorohexafluorocyclobutane, 2-chloroheptafluoropropane, and 2,3-chlorooctafluorobutane, did not affect GABAA receptor currents. Although all five compounds were predicted to be anesthetics by the Meyer Overton hypothesis, only F3 behaved as an anesthetic in vivo and only F3 markedly potentiated GABAA receptor responses in oocytes. These results strongly implicate the GABAA receptor in general anesthesia. Fluorescence polarization studies showed that anesthetics (enflurane and F3), but not nonasthetics (1,2 dichlorohexafluorocyclobutane and 2,3-chlorooctafluorobutane) disordered membrane lipids. Thus, for the compounds studied actions on both GABAA receptor function and lipid order distinguish between anesthetics and nonanesthetics.

Mice fed a saturated, Purina chow or unsaturated fat diet from birth have different synaptic membrane fatty acid compositions. The saturated diet decreased docosahexaenoic [22:6 (n - 3)] and increased eicosatrienoic [20:3 (n - 9)], docosatrienoic [22:3 (n - 9)] and docosapentaenoic [22:5 (n - 6)] fatty acids compared to the Purina chow or unsaturated fat diet. The most notable alterations occurred in the synaptic membrane phosphatidylserine and phosphatidylethanolamine fractions. The saturated diet also caused a minor increase in oleic acid [18:1 (n - 9)i1. Animals fed the saturated diet showed a small, significant decrease in nitrous oxide ED50 (dose required to abolish the righting reflex in half the animals) but not in isoflurane ED50, compared to animals fed the Purina chow or unsaturated fat diet. Thus, large alterations in several synaptic membrane fatty acid components have little or no influence on anesthetic requirement.

Mice exposed to 50% nitrous oxide for 3 weeks become tolerant to nitrous oxide. The nitrous oxide-tolerant animals are also tolerant to ethanol, as measured by a 40% decrease in ethanol-induced sleep times and 15% higher blood alcohol levels upon awakening compared to control mice raised in air. These results suggest a common mechanism for the development of tolerance to both alcohol and nitrous oxide.

Mice continuously exposed to a subanesthetic dose of nitrous oxide (N2O) become tolerant to nitrous oxide, as measured by an increase in their ED50 (dose required to abolish the righting reflex in 50% of animals) for N2O. The maximum increase in ED50 is approximately 0.25 atm for mice exposed to 40, 50 or 70% N2O for 2 to 3 weeks. Mice exposed to 25% N2O for 3 weeks do not develop tolerance. Mice exhibit a withdrawal syndrome when removed from the subanesthetic environment after exposure to 40 to 70% but not 25% N2O for 3 weeks. Tolerance to 50 or 70% N2O develops within 1 week and is maximal at 2 weeks. Tolerance to nitrous oxide is lost within 6 days following removal of the animals from the subanesthetic environment. Synaptic membrane fatty acid, phospholipid, and cholesterol compositions of tolerant animals are not significantly altered.