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.

Enhancement of high harmonics generated by field steering of electrons in a two-color orthogonally polarized laser field.

We demonstrate enhancement by 1 order of magnitude of the high-order harmonics generated in argon by combining a fundamental field at 1300 nm (10(14) W cm(-2)) and its orthogonally polarized second harmonic at 650 nm (2 × 10(13) W cm(-2)) and by controlling the relative phase between them. This extends earlier work by ensuring that the main effect is the combined field steering the electron trajectory with negligible contribution from multiphoton effects compared to the previous schemes with 800/400 nm fields. We access a broad energy range of harmonics (from 20 eV to 80 eV) at a low laser intensity (far below the ionization saturation limit) and observe deep modulation of the harmonic yield with a period of π in the relative phase. Strong field theoretical analysis reveals that this is principally due to the steering of the recolliding electron wave packet by the two-color field. Our modeling also shows that the atto chirp can be controlled, leading to production of shorter pulses.

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.

Indiscriminate coagulation screening of acute medical admissions: national cost ramifications.

BACKGROUND: Routine coagulation screening constitutes poor medical practice and is wasteful of resources. We aimed to determine the extent of inappropriate coagulopathy screening of acute medical admissions in a Scottish general hospital. METHODS: One hundred consecutive medical admissions were prospectively analysed, assessing whether or not a coagulation screen had been conducted on admission and whether or not this was indicated according to current hospital guidelines. Following targeted dissemination of guidelines to appropriate front door medical and nursing staff the audit was repeated. RESULTS: Pre-education, 58% of those for whom coagulation screening was not indicated were being tested. After targeted education, this figure was reduced to 32%. Preeducation, 81% of all patients in whom coagulation screening was indicated were tested. After targeted education, this figure was 86%. CONCLUSION: Indiscriminate coagulation screening is widespread amongst medical admissions to our unit. With simple targeted education, we reduced the rate of inappropriate testing by 26% without reducing the rate of appropriate testing. In a small district general hospital (where the mean local cost for processing a haematology specimen is 8.59 pounds) this translates into a saving of 21,000 pounds per annum. Extrapolated nationwide this represents a cost saving of 1.15 million pounds per annum in Scotland.

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.