Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABA(A) receptor.

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 ß3 subunits, we tested the hypothesis that etomidate acts through ß3-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 ß3-subunit (ß3-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 ß3-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, α5ß1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 µM and below), whereas α5ß2γ2L and α5ß3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5ß3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5ß2-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).

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.

Bidirectional modulation of isoflurane potency by intrathecal tetrodotoxin and veratridine in rats.

BACKGROUND AND PURPOSE: Results from several studies point to voltage-gated Na(+) channels as potential mediators of the immobility produced by inhaled anaesthetics. We hypothesized that the intrathecal administration of tetrodotoxin, a drug that blocks Na(+) channels, should enhance anaesthetic potency, and that concurrent administration of veratridine, a drug that augments Na(+) channel opening, should reverse the increase in potency. EXPERIMENTAL APPROACH: We measured the change in isoflurane potency for reducing movement in response to a painful stimulus as defined by MAC (minimum alveolar concentration of anaesthetic required to abolish movement in 50% of subjects) caused by intrathecal infusion of various concentrations of tetrodotoxin into the lumbothoracic subarachnoid space of rats, and the change in MAC caused by the administration of a fixed dose of tetrodotoxin plus various doses of intrathecal veratridine. KEY RESULTS: Intrathecal infusion of tetrodotoxin (0.078-0.63 microM) produced a reversible dose-related decrease in MAC, of more than 50% at the highest concentration. Intrathecal co-administration of veratridine (1.6-6.4 microM) reversed this decrease in a dose-related manner, with nearly complete reversal at the highest veratridine dose tested. CONCLUSIONS AND IMPLICATIONS: Intrathecal administration of tetrodotoxin increases isoflurane potency (decreases isoflurane MAC), and intrathecal administration of veratridine counteracts this effect in vivo. These findings are consistent with a role for voltage-gated Na(+) channel blockade in the immobility produced by inhaled anaesthetics.

Inhaled anesthesia: the original closed-loop drug administration paradigm.

We administer anesthetics to obtain therapeutic effects and minimize untoward side effects. Anesthetists can precisely control inhaled anesthetic concentrations by controlling end-tidal volatile anesthetic concentrations. This degree of control eliminates the need for closed-loop inhaled anesthetic systems. The low solubility of modern inhaled anesthetics adds to the stability and control of the anesthetic state; the effective inhaled concentration varies little during maintenance of anesthesia unless altered by the anesthetist. A less precise closed-loop system applies a processed electroencephalogram (EEG) to assess depth of anesthesia and enable accurate delivery of volatile and intravenous anesthetics to maintain a stable state of anesthesia.

Luciferase as a model for the site of inhaled anesthetic action.

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.

The convulsant and anesthetic properties of cis-trans isomers of 1,2-dichlorohexafluorocyclobutane and 1,2-dichloroethylene.

UNLABELLED: The differences in potencies of optical isomers of anesthetics support the hypothesis that anesthetics act by specific receptor interactions. Diastereoisomerism and geometrical isomerism offer further tests of this hypothesis but have not been explored. They are the subject of this report. We quantified the nonimmobilizing and convulsant properties of the cis and trans diastereomers of the nonimmobilizer 2N (1,2-dichlorohexafluorocyclobutane). Although the lipophilicity of the diastereomers predicts complete anesthesia at the partial pressures applied, neither diastereomer had anesthetic activity alone, and the cis form may have a small (10%) capacity to antagonize anesthesia, as defined by additive effects on the MAC (the minimum alveolar concentration required to suppress movement to a noxious stimulus in 50% of rats) of desflurane. Both diastereomers produced convulsions, the cis form being nearly twice as potent as the trans form: convulsant 50% effective dose (mean +/- SD) was 0.039 +/- 0.009 atmospheres (atm) for the purified cis and 0.064 +/- 0.009 atm for the purified trans isomer. The MAC value for cis-1,2-dichloroethylene equaled 0.0071 +/- 0.0006 atm, and MAC for trans-1,2-dichloroethylene equaled 0.0183 +/- 0.0031 atm. In qualitative accord with the Meyer-Overton hypothesis, the greater cis potency was associated with a greater lipophilicity. However, the product of MAC x solubility differed between the cis and trans isomers by 40%-50%. We conclude that neither the cis nor trans isomers of 2N have anesthetic properties, but isomerism does influence 2N's convulsant properties and the anesthetic properties of dichloroethylene. These isomeric effects may be as useful in defining receptor-anesthetic interactions as those found with optical isomers. IMPLICATIONS: Cis-trans isomerism can influence the convulsant properties of the nonimmobilizer 2N (1,2-dichlorohexafluorocyclobutane) and the anesthetic properties of dichloroethylene. Such isomeric effects may be as useful as those found with optical isomers in defining receptor-anesthetic interactions.

Age, minimum alveolar anesthetic concentration, and minimum alveolar anesthetic concentration-awake.

UNLABELLED: 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. IMPLICATIONS: 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.

The effect of rigidity, shape, unsaturation, and length on the anesthetic potency of hydrocarbons.

We previously hypothesized that anesthesia results from an action on two sites separated by 5 A. The hypothesis relied on the finding that fluorinated alkanes having active anesthetic sites at each end of the molecule produce anesthesia as long as the total number of carbon atoms in their structure does not exceed five (i.e., approximately 5 A), and on the sustaining of the 5-A separation by the rigidity produced by fluorination. In this study, we tested an alternative hypothesis: that the site of action cannot accommodate a rigid compound, particularly a rectilinear compound, having more than five carbon atoms, and that rigidity itself might limit the anesthetic potency of larger compounds. We tested the anesthetic potency of 11 hydrocarbons in which rigidity was increased by unsaturation. In 72 rats exposed to such compounds, we found that unsaturation, rigidity, or both produced by unsaturation either did not change (double bonds) or increased (triple bonds) potency for a given number of carbon atoms. For example, we found that the rectilinear, rigid 2,4-trans-trans-hexadiene was no less potent (minimum alveolar anesthetic concentration [MAC] 0.042 +/- 0.002 atm; mean +/- SD) than the flexible 1,5-hexadiene (0.047 +/- 0.005 atm) or n-hexane (0.0467 +/- 0.0055 atm) and that 3-hexyne was more potent (MAC 0.0146 +/- 0.0014 atm) than n-hexane (MAC 0.0467 +/- 0.0055 atm). We conclude that the site of anesthetic action can accommodate straight rigid structures of up to six carbons in length.

Both cerebral GABA(A) receptors and spinal GABA(A) receptors modulate the capacity of isoflurane to produce immobility.

We previously demonstrated that intrathecal administration of the noncompetitive gamma-aminobutyric acid type A (GABA(A)) receptor antagonist picrotoxin increased isoflurane MAC (the minimum alveolar concentration of anesthetic producing immobility in 50% of animals) by a maximum (ceiling effect) of approximately 40%. We also found that IV administration of picrotoxin increased MAC by more than 60%, without evidence of a ceiling effect. The larger increase with IV administration suggested a role of cerebral GABA(A) receptors. Accordingly, in this study we examined the effect of intracerebroventricular administration of picrotoxin in rats, finding that picrotoxin infusion into the third ventricle increased isoflurane MAC by a maximum of approximately 40%, without finding a ceiling effect. In addition, we concurrently infused picrotoxin into the intrathecal and intracerebroventricular spaces, producing an increase in MAC in excess of 70%, also with no evidence of a ceiling effect. The dose-response relationship for the intrathecal-intraventricular infusion paralleled that of the IV infusion but was shifted to the left by an order of magnitude. We conclude that both cerebral and spinal GABA(A) receptors modulate the capacity of inhaled anesthetics to produce immobility. Because other studies have shown that the spinal cord, and not the brain, mediates the capacity of inhaled anesthetics to produce immobility, these results call into question the relevance of GABA(A) receptors to the immobilizing action of isoflurane.

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 anesthetic potency of propanol and butanol versus propanethiol and butanethiol in alpha1 wild type and alpha1(S267Q) glycine receptors.

UNLABELLED: Although similar in shape and size, and although differing only by substitution of a sulfur atom for an oxygen atom, propanethiol and butanethiol differ markedly from propanol and butanol in their in vivo potency and physical properties. Recent theories of narcosis suggest that anesthetics may act by enhancing the effect of inhibitory agonists, such as glycine, on their receptors. We tested whether propanol, butanol, propanethiol, and butanethiol enhance the effect of glycine on alpha1 glycine receptors expressed in Xenopus laevis oocytes in a manner that reflects the in vivo differences found for potencies. As anticipated, we found an immediate parallel between in vivo (rat minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects) and in vitro (recombinant receptor) effects. All four compounds enhanced the effect of glycine on wild type receptors, and the extent of enhancement for a given minimum alveolar concentration-multiple was approximately the same for all compounds. We also found that propanethiol, butanethiol, propanol, and butanol did not affect, or minimally affected, the action of glycine in anesthetic resistant mutants in which the amino acid serine at position 267 was replaced by glutamine [alpha1(S267Q)]. IMPLICATIONS: The in vivo potencies of propanethiol, butanethiol, propanol, and butanol correlate with their capacities to enhance the effect of glycine on alpha1 glycine receptors expressed in Xenopus laevis oocytes. These results support the notion that a protein mediates anesthetic action.

The anesthetic potencies of alkanethiols for rats: relevance to theories of narcosis.

UNLABELLED: Meyer and Overton suggested that anesthetic potency correlates inversely with lipophilicity. Thus, MAC times the olive oil/gas partition coefficient equals an approximately constant value of 1.82 +/- 0.56 atm (mean +/- SD). MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. Although MAC times the olive oil/gas partition coefficient also equals an approximately constant value for normal alkanols from methanol through octanol, the value (0.156 +/- 0.072 atm) is 1/10th that found for conventional anesthetics. We hypothesized that substitution of sulfur for the oxygen in n-alkanols would decrease their saline/gas partition coefficients (i.e., decrease polarity) while sustaining lipid/gas partition coefficients. Further, we hypothesized that these changes would produce products of MAC times olive oil partition coefficients that approximate those of conventional anesthetics. To test these predictions, we measured MAC in rats, and saline and olive oil solubilities for the series H(CH(2))(n)SH, comparing the results with the series H(CH(2))(n)OH for compounds having three to six carbon atoms. As hypothesized, the alkanethiols had similar oil/gas partition coefficients, 1000-fold smaller saline gas partition coefficients, and MAC values 30 times greater than for comparable alkanols. Such findings are consistent with the notion that the greater potency of many alkanols (greater than would be predicted from conventional inhaled anesthetics and the Meyer-Overton hypothesis) results from their greater polarity. IMPLICATIONS: The in vivo anesthetic potency of alkanols and alkanethiols correlates with their lipophilicity and hydrophilicity.

Fractional distillation of acid contaminants from sevoflurane.

On two occasions, sevoflurane distributed for clinical practice has been found to be contaminated with compounds thought to include hydrogen fluoride (HF) and silicon tetrafluoride (SiF(4)). Both compounds can produce pulmonary injury. However, injury would require fractional distillation of the compounds during the course of sevoflurane vaporization. We hypothesized that such distillation would occur and that the compounds would vaporize more rapidly than would sevoflurane. Thus, we tested whether fractional distillation occurs during vaporization of sevoflurane containing HF or SiF(4), or from sevoflurane containing HF converted to other compounds by contact with glass. Vaporization of < 10% of the sevoflurane distilled 65%-99% of these compounds, SiF(4) distilling most rapidly, HF (converted to other acidic compounds, including SiF(4)) distilling nearly as rapidly, and HF slowest. Nuclear magnetic resonance studies indicated that HF interaction with glass changed all HF to three other compounds, one being SiF(4) and the others being unknown. HF and SiF4 distill from sevoflurane more rapidly than sevoflurane is vaporized. Measurement of acidity after sevoflurane administration may not reveal a previous presence of such contaminants.