Bernd Antkowiak

Molecular and neuronal substrates for general anaesthetics.

Although general anaesthesia has been of tremendous importance for the development of surgery, the underlying mechanisms by which this state is achieved are only just beginning to be understood in detail. In this review, we describe the neuronal systems that are thought to be involved in mediating clinically relevant actions of general anaesthetics, and we go on to discuss how the function of individual drug targets, in particular GABAA-receptor subtypes, can be revealed by genetic studies in vivo.

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.

Neocortex is the major target of sedative concentrations of volatile anaesthetics: strong depression of firing rates and increase of GABAA receptor-mediated inhibition

General anaesthetics cause sedation, amnesia and hypnosis. Although these clinically desired actions are indicative of an impairment of neocortical information processing, it is widely held that they are to a large part mediated by subcortical neural networks. Anaesthetic action on brain stem, basal forebrain and thalamus, all of which are known to modulate cortical excitability, would thus ultimately converge on neocortex, perturbing and reducing action potential activity therein. However, as neocortex harbours molecular targets of anaesthetics in high densities, notably GABAA receptors, neocortex itself should be very sensitive to anaesthetics. Here, we performed experiments to reveal the extent to which neocortex proper is a relevant target of the low concentrations of volatile anaesthetics causing sedation and hypnosis. We compared the effects of isoflurane, enflurane and halothane on spontaneous action potential activity of rat neocortical neurons in vivo and in isolated cortical networks in vitro, i.e. in the presence and absence of subcortical arousal systems. We observed that the anaesthetics decreased spontaneous firing of neurons via intracortical mechanisms; concentrations inducing hypnosis in humans reduced discharge rates both in vivo and in vitro to the same extent, approximately 50%. This decrease in neuronal activity was paralleled by a significant enhancement of neocortical GABAA receptor‐mediated inhibition. These findings challenge the notion of predominantly subcortical effects of volatile anaesthetics and suggest that intracortical targets, among them neocortical GABAA receptors, mediate the sedative and hypnotic properties of volatile anaesthetics.

Different actions of general anesthetics on the firing patterns of neocortical neurons mediated by the GABA(A) receptor.

BACKGROUND In cultured slice preparations of rat neocortical tissue, clinically relevant concentrations of volatile anesthetics mainly decreased action potential firing of neurons by enhancing gamma-aminobutyric acid (GABA(A)) receptor-mediated synaptic inhibition. The authors aim was to determine if other anesthetic agents are similarly effective in this model system and act via the same molecular mechanism. METHODS The actions of various general anesthetics on the firing patterns of neocortical neurons were investigated by extracellular single-unit recordings. RESULTS Pentobarbital, propofol, ketamine, and ethanol inhibited spontaneous action potential firing in a concentration-dependent manner. The estimated median effective concentration (EC50) values were close to or below the EC50 values for general anesthesia. Bath application of the GABA(A) antagonist bicuculline (100 microM) decreased the effectiveness of propofol, ethanol, halothane, isoflurane, enflurane, and diazepam by more than 90%, indicating that these agents acted predominantly via the GABA(A) receptor. The depressant effects of pentobarbital and ketamine were not significantly reduced by bicuculline treatment. Drugs acting mainly via the GABA(A) receptor altered the firing patterns of neocortical cells in different manners. Diazepam reduced the discharge rates by decreasing the number of action potentials per burst, leaving the burst rate unaffected. In contrast, muscimol, GABA, propofol, and volatile anesthetics decreased the burst rate. CONCLUSIONS Although several anesthetic agents acted nearly exclusively via the GABA(A) receptor, they changed the discharge patterns of cortical neurons in different ways. This finding is explained by GABA-mimetic or benzodiazepine-like molecular interactions.

Molecular and systemic mechanisms of general anaesthesia: the 'multi-site and multiple mechanisms' concept.

Purpose of review Amnesia, hypnosis and immobility are essential components of general anaesthesia. This review highlights recent advances in our understanding of how these components are achieved at a molecular level. Recent findings Commonly used volatile anaesthetic agents such as isoflurane or sevoflurane cause immobility by modulating multiple molecular targets predominantly in the spinal cord, including γ-aminobutyric acidA receptors, glycine receptors, glutamate receptors and TREK-1 potassium channels. In contrast, intravenously applied drugs such as propofol or etomidate depress spinal motor reflexes almost exclusively via enhancing γ-aminobutyric acidA receptor function. Studies on knock-in animals showed that etomidate and propofol act via γ-aminobutyric acidA receptors containing β3 subunits, whereas γ-aminobutyric acidA receptors including α2 and γ subunits mediate the myorelaxant properties of diazepam. These findings suggest that a large fraction of γ-aminobutyric acidA receptors in the spinal cord assemble from α2, β3 and most probably γ2 subunits. The hypnotic actions of etomidate are mediated by β3-containing γ-aminobutyric acidA receptors expressed in the brain. In contrast, γ-aminobutyric acidA receptors harbouring β2 subunits produce sedation, but not hypnosis. Furthermore, there is growing evidence that extrasynaptic γ-aminobutyric acidA receptors in the hippocampus containing α5 subunits contribute to amnesia. Summary Clinical anaesthesia is based on drug actions at multiple anatomical sites in the brain. The finding that amnesia, hypnosis and immobility involve distinct molecular targets opens new avenues for developing improved therapeutic strategies in anaesthesia.

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.

How do general anaesthetics work

Abstract. Almost a century ago, Meyer and Overton discovered a linear relationship between the potency of anaesthetic agents to induce general anaesthesia and their ability to accumulate in olive oil. Similar correlations between anaesthetic potency and lipid solubility were later reported from investigations on various experimental model systems. However, exceptions to the Meyer-Overton correlation exist in all these systems, indicating that lipid solubility is an important, but not the sole determinant of anaesthetic action. In the mammalian central nervous system, most general anaesthetics act at multiple molecular sites. It seems likely that not all of these effects are involved in anaesthesia. GABAA- and NMDA-receptor/ion channels have already been identified as relevant targets. However, further mechanisms, such as a blockade of Na+ channels and an activation of K+ channels, also come into play. A comparison of different anaesthetics seems to show that each compound has its own spectrum of molecular actions and thus shows specific, fingerprint-like effects on different levels of neuronal activity. This may explain why there is no known compound that specifically antagonises general anaesthesia. General anaesthesia is a multidimensional phenomenon. Unconsciousness, amnesia, analgesia, loss of sensory processing and the depression of spinal motor reflexes are important components. It was not realised until very recently that different molecular mechanisms might underlie these different components. These findings challenge traditional views, such as the assumption that one anaesthetic can be freely replaced by another.

Propofol and sevoflurane depress spinal neurons in vitro via different molecular targets.

Background:The capacity of general anesthetics to produce immobility is primarily spinally mediated. Recently, compelling evidence has been provided that the spinal actions of propofol involve &ggr;-aminobutyric acid type A (GABAA) receptors, whereas the contribution of glycine receptors remains uncertain. The relevant molecular targets of the commonly used volatile anesthetic sevoflurane in the spinal cord are largely unknown, but indirect evidence suggests a mechanism of action distinct from propofol. Methods:The effects of sevoflurane and propofol on spontaneous action potential firing were investigated by extracellular voltage recordings from ventral horn interneurons in cultured spinal cord tissue slices obtained from embryonic rats (embryonic days 14–15). Results:Propofol and sevoflurane reduced spontaneous action potential firing of neurons. Concentrations causing half-maximal effects (0.11 &mgr;m propofol, 0.11 mm sevoflurane) were lower than the median effective concentration immobility (1–1.5 &mgr;m propofol, 0.35 mm sevoflurane). At higher concentrations, complete inhibition of action potential activity was observed with sevoflurane but not with propofol. Effects of sevoflurane were mediated predominantly by glycine receptors (45%) and GABAA receptors (38%), whereas propofol acted almost exclusively via GABAA receptors (96%). Conclusions:The authors’ results suggest that glycine and GABAA receptors are the most important molecular targets mediating depressant effects of sevoflurane in the spinal cord. They provide evidence that sevoflurane causes immobility by a mechanism distinct from the actions of the intravenous anesthetic propofol. The finding that propofol acts exclusively via GABAA receptors can explain its limited capacity to depress spinal neurons in the authors’ study.

Effects of Small Concentrations of Volatile Anesthetics on Action Potential Firing of Neocortical Neurons In Vitro

Background Volatile general anesthetics depress neuronal activity in the mammalian central nervous system and enhance inhibitory Cl‐ currents flowing across the [Greek small letter gamma]‐aminobutyric acidA (GABA (A)) receptor ‐ ion channel complex. The extent to which an increase in GABAA‐mediated synaptic inhibition contributes to the decrease in neuronal firing must be determined, because many further effects of these agents have been reported on the molecular level. Methods The actions of halothane, isoflurane, and enflurane on the firing patterns of single neurons were investigated by extracellular recordings in organotypic slice cultures derived from the rat neocortex. Results Volatile anesthetics depressed spontaneous action potential firing of neocortical neurons in a concentration‐dependent manner. The estimated median effective concentration (EC50) values were about one half the EC50 values for general anesthesia. In the presence of the GABA (A) antagonist bicuculline (20 [micro sign]M), the effectiveness of halothane, isoflurane, and enflurane in reducing the discharge rates were diminished by 48 ‐ 65%, indicating that these drugs act via the GABAA receptor. Conclusions Together with recent investigations, our results provide evidence that halothane, isoflurane, and enflurane reduced spontaneous action potential firing of neocortical neurons in cultured brain slices mainly by increasing GABAA‐mediated synaptic inhibition. At concentrations, approximately one half the EC50 for general anesthesia, volatile anesthetics increased overall GABAA‐mediated synaptic inhibition about twofold, thus decreasing spontaneous action potential firing by half.

Cross-approximate entropy of cortical local field potentials quantifies effects of anesthesia - a pilot study in rats

BackgroundAnesthetics dose-dependently shift electroencephalographic (EEG) activity towards high-amplitude, slow rhythms, indicative of a synchronization of neuronal activity in thalamocortical networks. Additionally, they uncouple brain areas in higher (gamma) frequency ranges possibly underlying conscious perception. It is currently thought that both effects may impair brain function by impeding proper information exchange between cortical areas. But what happens at the local network level? Local networks with strong excitatory interconnections may be more resilient towards global changes in brain rhythms, but depend heavily on locally projecting, inhibitory interneurons. As anesthetics bias cortical networks towards inhibition, we hypothesized that they may cause excessive synchrony and compromise information processing already on a small spatial scale. Using a recently introduced measure of signal independence, cross-approximate entropy (XApEn), we investigated to what degree anesthetics synchronized local cortical network activity. We recorded local field potentials (LFP) from the somatosensory cortex of three rats chronically implanted with multielectrode arrays and compared activity patterns under control (awake state) with those at increasing concentrations of isoflurane, enflurane and halothane.ResultsCortical LFP signals were more synchronous, as expressed by XApEn, in the presence of anesthetics. Specifically, XApEn was a monotonously declining function of anesthetic concentration. Isoflurane and enflurane were indistinguishable; at a concentration of 1 MAC (the minimum alveolar concentration required to suppress movement in response to noxious stimuli in 50% of subjects) both volatile agents reduced XApEn by about 70%, whereas halothane was less potent (50% reduction).ConclusionsThe results suggest that anesthetics strongly diminish the independence of operation of local cortical neuronal populations, and that the quantification of these effects in terms of XApEn has a similar discriminatory power as changes of spontaneous action potential rates. Thus, XApEn of field potentials recorded from local cortical networks provides valuable information on the anesthetic state of the brain.