Mutations M287L and Q266I in the glycine receptor α1 subunit change sensitivity to volatile anesthetics in oocytes and neurons, but not the minimal alveolar concentration in knockin mice.

BACKGROUND: Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal glycine receptors with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement. METHODS: We produced single amino acid mutations into the glycine receptor α1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brainstem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice. RESULTS: Studies of glycine-evoked currents in brainstem neurons from knockin mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics. CONCLUSION: Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs.

Gamma-aminobutyric acid type A receptor ß3 subunit forebrain-specific knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: ß3 containing γ-aminobutyric acid type A receptors (GABA(A)-Rs) mediate behavioral end points of IV anesthetics such as immobility and hypnosis. A knockout mouse with targeted forebrain deletion of the ß3 subunit of the GABA(A)-R shows reduced sensitivity to the hypnotic effect of etomidate, as measured by the loss of righting reflex. The end points of amnesia and immobility produced by an inhaled anesthetic have yet to be evaluated in this conditional knockout. METHODS: We assessed forebrain selective ß3 conditional knockout mice and their littermate controls for conditional fear to evaluate amnesia and MAC, the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation, to assess immobility. Suppression of conditional fear was assessed for etomidate and isoflurane, and MAC was assessed for isoflurane. RESULTS: Etomidate equally suppressed conditional fear for both genotypes. The knockout showed resistance to the suppression of conditional fear produced by isoflurane in comparison with control littermates. Controls and knockouts did not differ in isoflurane MAC values. CONCLUSIONS: These results suggest that ß3 containing GABA(A)-Rs in the forebrain contribute to hippocampal-dependent memory suppressed by isoflurane, but not etomidate.

Ross C. Terrell, PhD, an anesthetic pioneer.

On December 30, 2010, Ross C. Terrell, PhD, died. With his passing at age 85, we lost one of the pioneers of modern anesthesia. Terrell synthesized most of the inhalation anesthetics used today, including desflurane, enflurane, isoflurane, and sevoflurane.

Slowing of the hippocampal θ rhythm correlates with anesthetic-induced amnesia.

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.

Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

Amygdala transcriptome and cellular mechanisms underlying stress-enhanced fear learning in a rat model of posttraumatic stress disorder.

Severe stress or trauma can cause permanent changes in brain circuitry, leading to dysregulation of fear responses and the development of posttraumatic stress disorder (PTSD). To date, little is known about the molecular mechanisms underlying stress-induced long-term plasticity in fear circuits. We addressed this question by using global gene expression profiling in an animal model of PTSD, stress-enhanced fear learning (SEFL). A total of 15 footshocks were used to induce SEFL and the volatile anesthetic isoflurane was used to suppress the behavioral effects of stress. Gene expression in lateral/basolateral amygdala was measured using microarrays at 3 weeks after the exposure to different combinations of shock and isoflurane. Shock produced robust effects on amygdalar transcriptome and isoflurane blocked or reversed many of the stress-induced changes. We used a modular approach to molecular profiles of shock and isoflurane and built a network of regulated genes, functional categories, and cell types that represent a mechanistic foundation of perturbation-induced plasticity in the amygdala. This analysis partitioned perturbation-induced changes in gene expression into neuron- and astrocyte-specific changes, highlighting a previously underappreciated role of astroglia in amygdalar plasticity. Many neuron-enriched genes were highly correlated with astrocyte-enriched genes, suggesting coordinated transcriptional responses to environmental challenges in these cell types. Several individual genes were validated using RT-PCR and behavioral pharmacology. This study is the first to propose specific cellular and molecular mechanisms underlying SEFL, an animal model of PTSD, and to nominate novel molecular and cellular targets with potential for therapeutic intervention in PTSD, including glycine and neuropeptide systems, chromatin remodeling, and gliotransmission.

Gamma-aminobutyric acid type A receptor alpha 4 subunit knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: General anesthesia produces multiple end points including immobility, hypnosis, sedation, and amnesia. Tonic inhibition via gamma-aminobutyric acid type A receptors (GABA(A)-Rs) may play a role in mediating behavioral end points that are suppressed by low concentrations of anesthetics (e.g., hypnosis and amnesia). GABA(A)-Rs containing the alpha4 subunit are highly concentrated in the hippocampus and thalamus, and when combined with delta subunits they mediate tonic inhibition, which is sensitive to low concentrations of isoflurane. METHODS: In this study, we used a GABA(A) alpha4 receptor knockout mouse line to evaluate the contribution of alpha4-containing GABA(A)-Rs to the effects of immobility, hypnosis, and amnesia produced by isoflurane. Knockout mice and their wild-type counterparts were assessed on 3 behavioral tests: conditional fear (to assess amnesia), loss of righting reflex (to assess hypnosis), and the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation in 50% of subjects (to assess immobility). RESULTS: Genetic inactivation of the alpha4 subunit reduced the amnestic effect of isoflurane, minimally affected loss of righting reflex, and had no effect on immobility. CONCLUSIONS: These results lend support to the hypothesis that different sites of action mediate different anesthetic end points and suggest that alpha4-containing GABA(A)-Rs are important mediators of the amnestic effect of isoflurane on hippocampal-dependent declarative memory.

Increasing the duration of isoflurane anesthesia decreases the minimum alveolar anesthetic concentration in 7-day-old but not in 60-day-old rats.

BACKGROUND: While studying neurotoxicity in rats, we observed that the anesthetic minimum alveolar anesthetic concentration (MAC) of isoflurane decreases with increasing duration of anesthesia in 7-day-old but not in 60-day-old rats. After 15 min of anesthesia in 7-day-old rats, MAC was 3.5% compared with 1.3% at 4 h. We investigated whether kinetic or dynamic factors mediated this decrease. METHODS: In 7-day-old rats, we measured inspired and cerebral partial pressures of isoflurane at MAC as a function of duration of anesthesia. In 60-day-old rats, we measured inspired partial pressures of isoflurane at MAC as a function of duration of anesthesia. Finally, we determined the effect of administering 1 mg/kg naloxone and of delaying the initiation of the MAC determination (pinching the tail) on MAC in 7-day-old rats. RESULTS: In 7-day-old rats, both inspired and cerebral measures of MAC decreased from 1 to 4 h. The inspired MAC decreased 56%, whereas the cerebral MAC decreased 33%. At 4 h, the inspired MAC approximated the cerebral MAC (i.e., the partial pressures did not differ appreciably). Neither administration of 1 mg/kg naloxone nor delaying tail clamping until 3 h reversed the decrease in MAC. In 60-day-old rats, inspired MAC of isoflurane was stable from 1 to 4 h of anesthesia. CONCLUSIONS: MAC of isoflurane decreases over 1-4 h of anesthesia in 7-day-old but not in 60-day-old rats. Both pharmacodynamic and a pharmacokinetic components contribute to the decrease in MAC in 7-day-old rats. Neither endorphins nor sensory desensitization mediate the pharmacodynamic component.

Isoflurane suppresses stress-enhanced fear learning in a rodent model of post-traumatic stress disorder.

BACKGROUND: A minority of patients who experience awareness and/or pain during surgery subsequently develop post-traumatic stress disorder. In a rodent model of post-traumatic stress disorder, stress-enhanced fear learning (SEFL), rats are preexposed to a stressor of 15 foot shocks. Subsequent exposure to a single foot shock produces an enhanced fear response. This effect is akin to sensitized reactions shown by some post-traumatic stress disorder patients to cues previously associated with the traumatic event. METHODS: The authors studied the effect of isoflurane and nitrous oxide on SEFL. Rats were exposed to the inhaled anesthetic during or after a 15-foot shock stressor. Then, rats were given a single foot shock in a different environment. Their fear response was quantified in response to the 15-foot shock and single-foot shock environments. SEFL longevity was tested by placing a 90-day period between the 15 foot shocks and the single foot shock. In addition, the intensity of the foot shock was increased to evaluate treatment effectiveness. RESULTS: Increasing isoflurane concentrations decreased SEFL when given during, but not after, the stressor. At 0.40 minimum alveolar concentration (MAC), isoflurane given during the stressor blocked SEFL 90 days later. A threefold increase in the stressor intensity increased the isoflurane concentration required to block SEFL to no more than 0.67 MAC. As with isoflurane, nitrous oxide suppressed SEFL at a similar MAC fraction. CONCLUSIONS: These results suggest that sufficient concentrations (perhaps 0.67 MAC or less) of an inhaled anesthetic may prevent SEFL.

A comparison of the molecular bases for N-methyl-D-aspartate-receptor inhibition versus immobilizing activities of volatile aromatic anesthetics.

BACKGROUND: Aromatic anesthetics exhibit a wide range of N-methyl-d-aspartate (NMDA) receptor inhibitory potencies and immobilizing activities. We sought to characterize the molecular basis of NMDA receptor inhibition using comparative molecular field analysis (CoMFA), and compare the results to those from an equivalent model for immobilizing activity. METHODS: Published potency data for 14 compounds were supplemented with new values for 2 additional agents. The anesthetics were divided into a training set (n = 12) used to formulate the activity models and a test set (n = 4) used to independently assess the models' predictive capability. The anesthetic structures were geometry optimized using ab initio quantum mechanics and aligned by field-fit minimization to provide the best correlation between the steric and electrostatic fields of the molecules and one or more lead structures. Orientations that yielded CoMFA models with the greatest predictive capability (assessed by leave-one-out cross-validation) were retained. RESULTS: The final CoMFA model for the inhibition of NR1/NR2B NMDA receptors explained 99.3% of the variance in the observed activities of the 12 training set agents (F(2,)(9) = 661.5, P < 0.0001). The model effectively predicted inhibitory potency for the training set (cross-validated r(2)(CV) = 0.944) and 4 excluded test set compounds (predictive r(2)(Pred) = 0.966). The equivalent model for immobility in response to noxious stimuli explained 98.0% of the variance in the observed activities for the training set (F(2,)(9) = 219.2, P < 0.0001) and exhibited adequate predictive capability for both the training set (r(2)(CV) = 0.872) and test set (r(2)(Pred) = 0.926) agents. Comparison of pharmacophoric maps showed that several key steric and electrostatic regions were common to both activity models, but differences were observed in the relative importance of these key regions with respect to the two aspects of anesthetic activity. CONCLUSIONS: The similarities in the pharmacophoric maps are consistent with NMDA receptors contributing part of the immobilizing activity of volatile aromatic anesthetics.

Obesity modestly affects inhaled anesthetic kinetics in humans.

BACKGROUND: Few studies have determined the effect of obesity on inhaled anesthetic pharmacokinetics. We hypothesized that the solubility of potent inhaled anesthetics in fat and increased body mass index (BMI) in obese patients interact to increase anesthetic uptake and decrease the rate at which the delivered (FD) and inspired (FI) concentrations of an inhaled anesthetic approach a constantly maintained alveolar concentration (end-tidal or FA). This hypothesis implies that the effect of obesity would be greater with a more soluble anesthetic such as isoflurane versus desflurane. METHODS: In 107 ASA physical status I-III patients, anesthesia was induced with propofol, tracheal intubation facilitated with neuromuscular blockade, and ventilation controlled with 50% nitrous oxide in oxygen to maintain end-tidal carbon dioxide concentrations between 35 and 45 mm Hg. Isoflurane or desflurane was administered in a 1 L/min inflow rate at FD concentrations sufficient to maintain FA at 0.6 minimum alveolar anesthetic concentration (0.7% or 3.7%, respectively). FD, FI, and FA were measured 5, 10, 20, 40, 60, 90, 120,150, and 180 min after starting potent inhaled anesthetic delivery. RESULTS: Fifty-nine patients received isoflurane and 48 received desflurane. BMI ranged between 18 and 63 kg/m(2) and demographic variables did not differ between anesthetic groups. For isoflurane, FD/FA or FI/FA weakly (but significantly) correlated with BMI at 9/18 time points whereas for desflurane FD/FA or FI/FA correlated significantly with BMI at only one time point (P < 0.01). After dividing each group into nonobese (BMI < 30) and obese (BMI > or = 30) patients, with isoflurane, FD/FA or FI/FA was higher in obese patients at four time points whereas there was no difference between nonobese and obese patients for desflurane. Patients receiving isoflurane took longer to respond to command after discontinuing anesthesia but obesity did not increase or decrease awakening time for either isoflurane or desflurane. When BMI was used to normalize FI/FA and FD/FA the median values for isoflurane consistently exceeded the median value for desflurane by factors ranging from 3 to 5, values comparable to the ratios of their blood/gas (3.1), muscle/gas (4.6), and fat/gas (5.4) partition coefficients. CONCLUSION: BMI modestly affects FD/FA and FI/FA, and this effect is most apparent for an anesthetic having a greater solubility in all tissues. An increased BMI increases anesthetic uptake and, thus, the need for delivered anesthetic to sustain a constant alveolar anesthetic concentration, particularly with a more soluble anesthetic. However, the increase with an increased body mass is small.

The effects of volatile aromatic anesthetics on voltage-gated Na+ channels expressed in Xenopus oocytes.

BACKGROUND: Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these anesthetics results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-D-aspartate (NMDA) receptor function and enhance gamma-amino butyric acid A receptor function, but these effects depend strongly on the chemical properties of the aromatic compounds. In the present study we tested whether diverse aromatic anesthetics consistently inhibit sodium channel function. METHODS: We studied the effect of eight aromatic anesthetics on Na(v)1.2 sodium channels with beta(1) subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. RESULTS: All aromatic anesthetics inhibited I(Na) (sodium currents) at a holding potential which produce half-maximal current (V(1/2)) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8% +/- 2%), pentafluorobenzene (13% +/- 2%), and hexafluorobenzene (13% +/- 2%), but greater with benzene (37% +/- 2%), fluorobenzene (39% +/- 2%), 1,2-difluorobenzene (48% +/- 2%), 1,4-difluorobenzene (31 +/- 3%), and 1,2,4-trifluorobenzene (33% +/- 1%). Such dichotomous effects were noted by others for NMDA and gamma-aminobutyric acid A receptors. Parallel, but much smaller inhibition, was found for I(Na) at a holding potential which produced near maximal current (-90 mV) (V(H-90)), and hexafluorobenzene caused small (6% +/- 1%) enhancement of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-pi interactions. CONCLUSION: Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by minimum alveolar anesthetic concentration concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the action of some of these anesthetics, but not others.