Berthold Drexler

General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit.

General anesthetics are widely used in clinical practice. On the molecular level, these compounds have been shown to modulate the activity of various neuronal ion channels. However, the functional relevance of identified sites in mediating essential components of the general anesthetic state, such as immobility and hypnosis, is still unknown. Using gene‐targeting technology, we generated mice harboring a subtle point mutation (N265M) in the second transmembrane region of the β3 subunit of the GABAA receptor. In these mice, the suppression of noxious‐evoked movements in response to the intravenous anesthetics etomidate and propofol is completely abolished, while only slightly decreased with the volatile anesthetics enflurane and halothane. β3(N265M) mice also display a profound reduction in the loss of righting reflex duration in response to intravenous but not volatile anesthetics. In addition, electrophysiological recordings revealed that anesthetic agents were significantly less effective in enhancing GABAA receptor‐mediated currents, and in decreasing spontaneous action potential firing in cortical brain slices derived from mutant mice. Taken together, our results demonstrate that a single molecular target, and indeed a specific residue (N265) located within the GABAA receptor β3 subunit, is a major determinant of behavioral responses evoked by the intravenous anesthetics etomidate and propofol, whereas volatile anesthetics appear to act via a broader spectrum of molecular targets.

Anaesthetic drugs: linking molecular actions to clinical effects.

The use of general anaesthetics has facilitated great advantages in surgery within the last 150 years. General anaesthesia is composed of several components including analgesia, amnesia, hypnosis and immobility. To achieve these components, general anaesthetics have to act via multiple molecular targets at different anatomical sites in the central nervous system. Much of our current understanding of how anaesthetics work has been obtained within the last few years on the basis of genetic approaches, in particular knock-out or knock-in mice. Anaesthetic drugs can be grouped into volatile and intravenous anaesthetics according to their route of administration. Common volatile anaesthetics induce immobility via molecular targets in the spinal cord, including glycine receptors, GABA(A) receptors, glutamate receptors, and TREK-1 potassium channels. In contrast, intravenous anaesthetics cause immobility almost exclusively via GABA(A) receptors harbouring beta3 subunits. Hypnosis is predominantly mediated by beta3-subunit containing GABA(A) receptors in the brain, whereas beta2 subunit containing receptors, which make up more than 50% of all GABA(A) receptors in the central nervous system, mediate sedation. At clinically relevant concentrations, ketamine and nitrous oxide block NMDA receptors. Unlike all other anaesthetics in clinical use they produce analgesia. Not only desired actions of anaesthetics, but also undesired side effects are linked to certain receptors. Respiratory depression involves beta3 containing GABA(A) receptors whereas hypothermia is largely mediated by GABA(A) receptors containing beta2 subunits. These recent insights into the clinically desired and undesired actions of anaesthetic agents provide new avenues for the design of drugs with an improved side-effect profile. Such agents would be especially beneficial for the treatment of newborn children, elderly patients and patients undergoing ambulatory surgery.

Inhibitory Ligand-Gated Ion Channels as Substrates for General Anesthetic Actions

General anesthetics have been in clinical use for more than 160 years. Nevertheless, their mechanism of action is still only poorly understood. In this review, we describe studies suggesting that inhibitory ligand-gated ion channels are potential targets for general anesthetics in vitro and describe how the involvement of y-aminobutyric acid (GABA)(A) receptor subtypes in anesthetic actions could be demonstrated by genetic studies in vivo.

Opposing Actions of Etomidate on Cortical Theta Oscillations Are Mediated by Different γ-Aminobutyric Acid Type A Receptor Subtypes

Background:Cortical networks generate diverse patterns of rhythmic activity. Theta oscillations (4–12 Hz) are commonly observed during spatial learning and working memory tasks. The authors ask how etomidate, acting predominantly via &ggr;-aminobutyric acid type A (GABAA) receptors containing &bgr;2 or &bgr;3 subunits, affects theta activity in vitro. Methods:To characterize the effects of etomidate, the authors recorded action potential firing together with local field potentials in slice cultures prepared from the neocortex of the &bgr;3(N265M) knock-in mutant and wild type mice. Actions of etomidate were studied at 0.2 &mgr;m, which is approximately 15% of the concentration causing immobility (∼1.5 &mgr;m). Results:In preparations derived from wild type and &bgr;3(N265M) mutant mice, episodes of ongoing activity spontaneously occurred at a frequency of approximately 0.1 Hz and persisted for several seconds. Towards the end of these periods, synchronized oscillations in the theta band developed. These oscillations were significantly depressed in slices from &bgr;3(N265M) mutant mice (P < 0.05). In this preparation etomidate acts almost exclusively via &bgr;2 subunit containing GABAA receptors. In contrast, no depression was observed in slices from wild type mice, where etomidate potentiates both &bgr;2- and &bgr;3-containing GABAA receptors. Conclusions:At concentrations assumed to cause sedation and amnesia, etomidate depresses theta oscillations via &bgr;2-containing GABAA receptors but enhances these oscillations by acting on &bgr;3 subunit containing receptors. This indicates that the overall effect of the anesthetic reflects a balance between enhancement and inhibition produced by different GABAA receptor subtypes.

Distinct actions of etomidate and propofol at β3-containing γ-aminobutyric acid type A receptors

Etomidate and propofol have clearly distinguishable effects on the central nervous system. However, studies in knock-in mice provided evidence that these agents produce anesthesia via largely overlapping molecular targets, namely GABA(A) receptors containing beta3 subunits. Here the authors address the question as to whether etomidate and propofol are targeting different subpopulations of beta3 subunit containing GABA(A) receptors. The effects of etomidate and propofol (0.5 muM and 1.0 muM) on spontaneous activity of neocortical neurons were investigated in organotypic slice cultures from wild-type and beta3(N265M) knock-in mice. Firing patterns were characterized by mean burst length and number of action potentials per burst. Additionally, etomidate and propofol actions on GABA(A) receptor-mediated currents were investigated by whole-cell voltage clamp recordings. On the network level, the duration of spontaneously occurring bursts of action potentials was decreased by etomidate but increased by propofol in the wild-type. The effects of etomidate were abolished in beta3(N265M) mutant slices while those of propofol were qualitatively inverted. On the receptor level, GABA(A) receptor-mediated inhibition of cortical neurons was modulated by etomidate and propofol in different ways. Again, drug-specific actions of etomidate and propofol were largely attenuated by the beta3(N265M) mutation. Etomidate and propofol alter the firing patterns and GABA(A) receptor-mediated inhibition of neocortical neurons in different ways. This suggests that etomidate and propofol act via non-uniform molecular targets. Because the major effects induced by these anesthetics were attenuated by the beta3(N265M) mutation, different subpopulations of beta3-containing GABA(A) receptors are likely to be involved.

Cholinergic Modulation of Sevoflurane Potency in Cortical and Spinal Networks In Vitro

Background:Victims of organophosphate intoxication with cholinergic crisis may have need for sedation and anesthesia, but little is known about how anesthetics work in these patients. Recent studies suggest that cholinergic stimulation impairs &ggr;-aminobutyric acid type A (GABAA) receptor function. Because GABAA receptors are major targets of general anesthetics, the authors investigated interactions between acetylcholine and sevoflurane in spinal and cortical networks. Methods:Cultured spinal and cortical tissue slices were obtained from embryonic and newborn mice. Drug effects were assessed by extracellular voltage recordings of spontaneous action potential activity. Results:Sevoflurane caused a concentration-dependent decrease in spontaneous action potential firing in spinal (EC50 = 0.17 ± 0.02 mm) and cortical (EC50 = 0.29 ± 0.01 mm) slices. Acetylcholine elevated neuronal excitation in both preparations and diminished the potency of sevoflurane in reducing action potential firing in cortical but not in spinal slices. This brain region-specific decrease in sevoflurane potency was mimicked by the specific GABAA receptor antagonist bicuculline, suggesting that (1) GABAA receptors are major molecular targets for sevoflurane in the cortex but not in the spinal cord and (2) acetylcholine impairs the efficacy of GABAA receptor–mediated inhibition. The latter hypothesis was supported by the finding that acetylcholine reduced the potency of etomidate in depressing cortical and spinal neurons. Conclusions:The authors raise the question whether cholinergic overstimulation decreases the efficacy of GABAA receptor function in patients with organophosphate intoxication, thereby compromising anesthetic effects that are mediated predominantly via these receptors such as sedation and hypnosis.

Diazepam decreases action potential firing of neocortical neurons via two distinct mechanisms.

BACKGROUND: Benzodiazepines are widely used in clinical anesthesia as premedication, but also to induce general anesthesia. Recent in vitro studies suggest that &ggr;-aminobutyric acid type A receptors, harboring a classical high-affinity benzodiazepine binding site, possess another “nonclassical” binding site for benzodiazepines. At present, it is unclear if, and to what extent, this novel nonclassical binding site is of relevance for the actions of benzodiazepines in the central nervous system. METHODS: Because neocortex is involved in mediating the sedative and hypnotic properties of general anesthetics, we quantified the actions of diazepam over a wide range of concentrations (from 10 nM up to 100 &mgr;M) in organotypic slice cultures using extracellular multiunit recordings of spontaneous action potential activity. RESULTS: Up to a concentration of 6.25 &mgr;M, diazepam reduced the activity of neocortical neurons, approaching a maximum of approximately 20%. This action was nullified by the benzodiazepine antagonist flumazenil. At concentrations >12.5 &mgr;M, diazepam evoked a second concentration-dependent dampening of network activity. Unlike the low concentration effect, this high concentration component was resistant to flumazenil. CONCLUSIONS: Diazepam induced a biphasic attenuation of spontaneous action potential firing of neocortical neurons. Low to moderate concentrations caused a monotonic, mild depression that is mediated via the classical binding site as it is antagonized by flumazenil. However, the effects of diazepam observed at high concentrations were not affected by flumazenil. Hence, these findings support the concept of at least 2 different binding sites for benzodiazepines on &ggr;-aminobutyric acid type A receptors. Furthermore, our results are consistent with the hypothesis that the classical high-affinity binding site mediates low-dose diazepam actions, such as amnesia, anxiolysis, and sedation, while a second, nonclassical and independent site contributes to the anesthetic effects of diazepam, such as hypnosis and immobility.

Dual Actions of Enflurane on Postsynaptic Currents Abolished by the γ-Aminobutyric Acid Type A Receptor β3(N265M) Point Mutation

Background:At concentrations close to 1 minimum alveolar concentration (MAC)-immobility, volatile anesthetics display blocking and prolonging effects on γ-aminobutyric acid type A receptor-mediated postsynaptic currents. It has been proposed that distinct molecular mechanisms underlie these dual actions. The authors investigated whether the blocking or the prolonging effect of enflurane is altered by a point mutation (N265M) in the β3 subunit of the γ-aminobutyric acid type A receptor. Furthermore, the role of the β3 subunit in producing the depressant actions of enflurane on neocortical neurons was elucidated. Methods:Spontaneous inhibitory postsynaptic currents were sampled from neocortical neurons in cultured slices derived from wild-type and β3(N265M) mutant mice. The effects of 0.3 and 0.6 mm enflurane on decay kinetics, peak amplitude, and charge transfer were quantified. Furthermore, the impact of enflurane-induced changes in spontaneous action potential firing was evaluated by extracellular recordings in slices from wild-type and mutant mice. Results:In slices derived from wild-type mice, enflurane prolonged inhibitory postsynaptic current decays and decreased peak amplitudes. Both effects were almost absent in slices from β3(N265M) mutant mice. At clinically relevant concentrations between MAC-awake and MAC-immobility, the anesthetic was less effective in depressing spontaneous action potential firing in slices from β3(N265M) mutant mice compared with wild-type mice. Conclusion:At concentrations between MAC-awake and MAC-immobility, β3-containing γ-aminobutyric acid type A receptors contribute to the depressant actions of enflurane in the neocortex. The β3(N265M) mutation affects both the prolonging and blocking effects of enflurane on γ-aminobutyric acid type A receptor-mediated inhibitory postsynaptic currents in neocortical neurons.

Identification and characterization of anesthetic targets by mouse molecular genetics approaches

PurposeIt is now generally accepted that proteins are the primary targets of general anesthetics. However, the demonstration that the activity of a protein is altered by general anesthetics at clinically relevant concentrations in vitro does not provide direct evidence that this target mediates pharmacological actions of general anesthetics. Here we report on advances that have been made in identifying the contribution of individual ligand-gated ion channels to defined anesthetic endpoints using molecular mouse genetics.Principal findingsGamma-aminobutyric acid (GABA)A receptor subtypes defined by the presence of the α1, α4, α5, β2, and β3 subunits and two-pore domain potassium channels (TASK-1, TASK-3, and TREK) have been discovered to mediate, at least in part, the hypnotic, immobilizing or amnestic actions of intravenous and volatile general anesthetics. Moreover, using tissues from genetically modified mice, specific functions of GABAA receptor subtypes in cortical and spinal neuronal networks were identified.ConclusionGenetically modified mice have been very useful for research on mechanisms of anesthesia and have contributed to the functional identification of general anesthetic targets and of the role of these targets in neuronal networks.RésuméObjectifAujourd’hui, il est universellement accepté que les protéines sont les cibles principales des anesthésiques généraux. Cependant, la démonstration que l’activité d’une protéine est modifiée in vitro par les anesthésiques généraux en concentrations pertinentes d’un point de vue clinique ne constitue pas une donnée probante directe prouvant que cette cible est le médiateur des actions pharmacologiques des anesthésiques généraux. Nous rapportons ici les progrès accomplis dans la détermination de la contribution des canaux ioniques individuels sensibles à un ligand à la définition des critères anesthésiques en utilisant la génétique moléculaire de la souris.Constatations principalesOn a découvert que les sous-types de récepteurs GABAA tels que définis par la présence des sous-unités α1, α4, α5, β2, et β3 et les canaux potassiques à deux domaines P (TASK-1, TASK-3 et TREK) médiaient au moins partiellement les actions hypnotiques, immobilisantes ou amnésiques des anesthésiques généraux intraveineux ou volatils. De plus, en analysant des tissus de souris génétiquement modifiées, nous avons pu identifier certaines fonctions spécifiques des sous-types de récepteurs GABAA dans les réseaux neuronaux corticaux et rachidiens.ConclusionLes souris génétiquement modifiées ont été très utiles à la recherche sur les mécanismes de l’anesthésie et ont contribué à l’identification fonctionnelle des cibles des anesthésiques généraux et du rôle de ces cibles dans les réseaux neuronaux.

Long-term evaluation of organophosphate toxicity and antidotal therapy in co-cultures of spinal cord and muscle tissue ☆

Victims of nerve agents basically require antidotal treatment. There is need for novel antidotes and for therapeutic procedures that are specifically adapted to these patients. To cope with this challenge, in vitro test systems which are easy to handle and allow for conducting long-term studies would be of great benefit. The present work introduces co-cultures of spinal cord and muscle tissue as ex vivo testing systems meeting these criteria. Cell cultures in which functional neuromuscular synapses formed ex vivo were prepared from embryonic mice. Spontaneous muscle activity was recorded by video microscopy. Muscle contractions involved intact neuromuscular transmission as indicated by the effect of succinylcholine, a muscle relaxant that completely abolished muscle activity. At a concentration of 0.75 μM the nerve agent VX reduced the frequency of spontaneous muscle contractions by about 75%. Subsequent application of obidoxime re-established muscle movements. After 24 h of antidotal treatment, muscle activity approached the level of sham-treated cultures and remained stable over the following week. In summary, co-cultures of spinal cord and muscle tissue are promising tools for evaluating the success of antidotal treatment following organophosphate intoxication over a period of at least seven days.