Closing the gap between the molecular and systemic actions of anesthetic agents.

CHAPTER NINE

Closing the Gap Between the Molecular and Systemic Actions of Anesthetic Agents Bernd Antkowiak*,†,1 *Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology Section, Eberhard-Karls-University, T€ ubingen, Germany † Werner Reichardt Centre for Integrative Neuroscience, Eberhard-Karls-University, T€ ubingen, Germany 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction Classical Theories of General Anesthesia Point Mutations in GABAA Receptors Affecting Anesthetic Potency Neuroanatomical Substrates for General Anesthetics Homeostatic Regulations in Knockout Animals Anesthetic-Resistant Mice The Hypnotic Action of Etomidate Etomidate-Induced Hypnosis and Subtype-Specific Electroencephalogram Signatures 9. Benzodiazepine-Induced Sedation Does Not Manifest in the EEG 10. Different Roles of α2- and α3-Subunits in Modulating Brain Electrical Activity 11. Intracortical Actions of Etomidate 12. Actions of Etomidate in the Hippocampus 13. Spinal Actions of Etomidate 14. Anesthetic Side Effects 15. Multisite and Multiple Molecular Actions of General Anesthetics 16. Agent-Specific Actions of Anesthetics Lacking Binding Selectivity 17. Conclusion Conflict of Interest Acknowledgments References

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Abstract Genetic approaches have been successfully used to relate the diverse molecular actions of anesthetic agents to their amnestic, sedative, hypnotic, and immobilizing properties. The hypnotic effect of etomidate, quantified as the duration of the loss of righting reflex in mice, is equally mediated by GABAA receptors containing β2- and β3-protein subunits. However, only β3-containing receptors are involved in producing Advances in Pharmacology, Volume 72 ISSN 1054-3589 http://dx.doi.org/10.1016/bs.apha.2014.10.009

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2015 Elsevier Inc. All rights reserved.

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electroencephalogram (EEG)-patterns typical of general anesthesia. The sedative action of diazepam is produced by α1-subunit-containing receptors, but these receptors do not contribute to the drug's characteristic EEG-“fingerprint.” Thus, GABAA receptors with α1- and β2-subunits take a central role in causing benzodiazepine-induced sedation and etomidate-induced hypnosis, but the corresponding EEG-signature is difficult to resolve. Contrastingly, actions of etomidate and benzodiazepines mediated via α2- and β3subunits modify rhythmic brain activity in vitro and in vivo at least in part by enhancing neuronal synchrony. The immobilizing action of GABAergic anesthetics predominantly involves β3-subunit-containing GABAA receptors in the spinal cord. Interestingly, this action is self-limiting as GABA-release is attenuated via the same receptors. Anesthetic-induced amnesia is in part mediated by GABAA receptors harboring α5-subunits that are highly enriched in the hippocampus and, in addition, by α1-containing receptors in the forebrain. Because there is accumulating evidence that in patients the expression pattern of GABAA receptor subtypes varies with age, is altered by the long-term use of drugs, and is affected by pathological conditions like inflammation and sepsis, further research is recommended to adapt the use of anesthetic agents to the specific requirements of individual patients.

ABBREVIATIONS CNS central nervous system EEG electroencephalogram GABA γ-aminobutyric acid IPSC inhibitory postsynaptic currents LFP local field potential NMDA N-methyl-D-aspartic acid

1. INTRODUCTION Our understanding of the molecular and cellular mechanisms causing general anesthesia improved considerably during the past decades. Chemical structures of general anesthetic agents display great diversity (Urban, Bleckwenn, & Barann, 2006). However, these agents have in common to strongly decrease the excitability and activity of neurons in the central nervous system (CNS). It has long been thought that these compounds do not act via specific receptors. Yet, the discovery that general anesthetics bind to proteins changed this view (Ueda, 1965; Ueda & Kamaya, 1973). In search of the molecular mechanism underlying general anesthesia, scientists focused on ion channels sited on central neurons. A large number of in vitro studies provided evidence that at clinically relevant concentrations, a single

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anesthetic agent alters the function of multiple neuronal ion channels (Campagna, Miller, & Forman, 2003). But how do we find out which of the possible molecular targets do in fact contribute to anesthesia? The explanatory gap between molecular drug actions characterized in vitro and their unknown role in vivo was a long-standing problem. In a first attempt to answer the question what interactions between anesthetic agents and ion channels translate into clinically relevant effects including sedation, unconsciousness, amnesia or immobility, knockout mice were created lacking potential anesthetic targets (Homanics et al., 1997). However, in several cases studies on knockout mice provided ambiguous results. Fortunately, this Gordian knot was cut by Hanns M€ ohler’s group. The scientific community rated the successful development of anesthetic-resistant knockin mice as a milestone in their field of studies, similar in importance to the discovery of the Meyer–Overton correlation and the finding that anesthetic agents act on proteins (Harrison, 2003). Because GABAA receptors are a major molecular target for general anesthetic agents (Rudolph & Antkowiak, 2004), Hanns M€ ohler’s research on benzodiazepines is highly recognized by investigators exploring the molecular basis of general anesthesia. This chapter reviews the progress made in our understanding how anesthetic agents work. In particular, it shines light on the lessons learned from anesthetic-resistant and benzodiazepine-resistant knockin mice invented by Hanns M€ ohler’s group ( Jurd et al., 2003; Kopp, Rudolph, Low, & Tobler, 2004; Rudolph et al., 1999). First, classical concepts and theories of general anesthesia are shortly reviewed. Thereafter, pioneering studies on the interactions between general anesthetics and point mutated GABAA receptors are highlighted. Furthermore, γ-aminobutyric acid (GABA)-receptor heterogeneity and insights into the actions of etomidate as provided by studies on knockin mice are discussed. These findings are related to work on benzodiazepine-insensitive knockin mice. Furthermore, the striking similarities between Hanns M€ ohler’s new benzodiazepine pharmacology and the “multisite and multiple-mechanisms” concept of anesthetic action are discussed. In Section 17, I consider how these novel findings may translate into strategies to improve clinical anaesthesia. This chapter does not provide a comprehensive overview on the molecular mechanisms that are involved in general anesthesia. Rather, studies are highlighted that are closely related to or strongly influenced by Hanns M€ ohler’s work.

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2. CLASSICAL THEORIES OF GENERAL ANESTHESIA In 1846, the Boston dentist William T. Morton successfully demonstrated how to use ether for anesthetizing a patient who underwent surgery. Up to this date, surgery was almost exclusively performed on fully conscious subjects, frequently crying out in pain during the operation. Because painful surgical incisions cause reflex movements, these patients had to be mechanically fixed to facilitate surgical interventions. Keeping in mind how surgery was performed before anesthesia was invented, it is easy to understand that Morton’s presentation was highly appreciated by patients and surgeons and, as a consequence, the use of anesthetic agents rapidly developed into a gold standard. Already in these early days of general anesthesia, several volatile agents with diverging chemical structures were around for proving amnesia, unconsciousness, and immobility. Thus the question emerged as to how these different compounds cause the same effect in patients, namely general anesthesia. Around the year 1900, the pharmacologists Meyer and Overton independently discovered that the potency of anesthetic drugs in producing anesthesia in tadpoles well correlated with their ability to accumulate in olive oil (Overton, 1901). This correlation, later called the Meyer–Overton correlation, prompted scientists to offer reasonable explanations. For example, Meyer’s theory of anesthetic action supposed that anesthetic agents accumulate into the lipid bilayer that surrounds nerve cells (Meyer, 1937). It assumed that anesthesia happens once a critical concentration of anesthetic compounds is reached in the nerve membrane. Because trapping of anesthetic agents within biological membranes is positively correlated with the hydrophobicity of the respective compounds, anesthetic potency is determined by lipid solubility. But how to explain that the different chemical structures of anesthetic agents do not matter? It was reasoned that the presence of small hydrophobic molecules in the lipid membrane alters the membrane’s chemophysical properties irrespective of the drug’s chemical structure. Moreover, it was thought that altered chemophysical properties of the lipid bilayer indirectly, i.e., without direct interactions between anesthetic compounds and transmembrane proteins, translate into an altered function of ion channels. As a consequence of this mechanism, excitability of central neurons and their capacity to store and process information decreases and general anesthesia happens. At this point, it is important to clearly distinguish between the Meyer–Overton correlation as a phenomenological description of drug properties and molecular theories of anesthetic action, trying to explain this correlation. The Meyer–Overton rule applies,


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with a few exceptions, to anesthetic actions observed in many different biological systems (Urban et al., 2006). In these systems, the rule applies to different levels of complexity including ion channels, cells, neuronal networks, or reflex behavior. However, the assumption that interactions between proteins and general anesthetic agents are mostly unspecific turned out to be wrong. In our days, there is ample evidence that lipid solubility is only one determinant of anesthetic potency among others. In 1965, Ueda reported that a water-soluble enzyme, the firefly luciferase, interacts with clinically relevant concentrations of volatile anesthetics (Ueda, 1965). Most important, the efficacy of a number of anesthetic agents in inhibiting the chemical reaction catalyzed by the firefly luciferase was in accordance with the prediction of the Meyer–Overton rule (Franks & Lieb, 1984; Ueda & Kamaya, 1973). These studies formed a core of results leading to the protein theory of anesthetic action. In 1973, Eyring and coworkers wrote: “The basic idea is that anesthetic molecules combine with hydrophobic regions of protein or proteins essential to the maintenance of consciousness, thereby forming a conformational change to a less active, or an inactive form” (Eyring, Woodbury, & D’Arrigo, 1973). Some years later Franks and Lieb showed that at clinically relevant concentrations general anesthetic agents do not produce structural changes of lipid bilayers as postulated by the lipid theory (Franks & Lieb, 1978, 1982). Of course, the luciferase reaction does not play an important role in the CNS. However, the finding that a water-soluble protein is sensitive to anesthetic drugs opened up new perspectives and prompted many questions. As a consequence, scientific discussions now focused on the topic what ion channels might be involved and how many molecular targets are contributing to the anesthetic state. In these days, many different molecular targets, mostly ion channels, were proposed to play a key role in anesthesia. They include voltage-dependent sodium channels, potassium channels, glutamate, GABA, and glycine receptors among others (Campagna et al., 2003). Furthermore, the number of molecular targets involved in anesthesia was debated controversially. Experts’ opinions ranged between a single protein, a few relevant targets and thousands of actions that need to sum up for producing anesthesia (Eckenhoff & Johansson, 1999; Flohr, Glade, & Motzko, 1998; Franks & Lieb, 1993). Moreover, the range of effective concentrations and the required effect size of altered ion channel function necessary to cause anesthesia were disputed (Eger et al., 2001). However, the clinical significance of the proposed theories remained a matter of belief. At that time, the relevance of suggested targets in clinical anesthesia, identified by in vitro studies, could not be tested experimentally.

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3. POINT MUTATIONS IN GABAA RECEPTORS AFFECTING ANESTHETIC POTENCY GABAA receptors were early identified as potential targets for general anesthetics (Tanelian, Kosek, Mody, & MacIver, 1993). These receptors are heteropentamers, assembling from 19 known protein subunits (α1–6, β1–3, γ1–3, δ, ε, π, ρ1–3). Approximately 60% of all GABAA receptors are built from α1, β2, and γ2 subunits, 15–20% show an α2, β3, γ2 combination, and 10–15% have an α3, βn, γ2 combination (Olsen & Sieghart, 2008; Whiting, 2003). There are two binding sites for the agonist on each receptor formed by parts of an α- and a β-subunit (Kash, Trudell, & Harrison, 2004). GABAA receptors can be located at synapses or extrasynaptically, mediating phasic or tonic inhibition (Mody & Pearce, 2004). Agonist-induced activation of GABAA receptors causes these ion channels to open, allowing anions to flow into cells, thereby reducing the excitability of the respective neuron. GABA-induced activation of GABAA receptors is potentiated by most anesthetic agents in current use (Grasshoff, Drexler, Rudolph, & Antkowiak, 2006). Thus, the presence of anesthetics augments the neurotransmitter’s impact to reduce neuronal excitability. Moreover, actions of different anesthetic agents on GABAA receptors are consistent with the predictions of the Meyer–Overton rule (Zimmerman, Jones, & Harrison, 1994). These properties suggested GABAA receptors to be major players in general anesthesia. Studies on insecticide-resistant mutants of Drosophila melanogaster strongly influenced research into anesthetic mechanisms. Cyclodiene insecticides act as antagonists at GABAA receptors. Interestingly, a small subpopulation of Drosophila (the Drosophila mutant RDL) proved to be resistant against the insecticide dieldrine. In these mutants, Ffrench-Constant and coworkers identified a resistance-associated point mutation that strongly affected the binding of cyclodiene insecticides to GABAA receptors (Ffrench-Constant, Mortlock, Shaffer, MacIntyre, & Roush, 1991). Based on this work, Belelli and colleagues wondered whether alteration of a single amino acid may also affect the binding of general anesthetic agents to GABAA receptors (Belelli, Callachan, Hill-Venning, Peters, & Lambert, 1996; McGurk, Pistis, Belelli, Hope, & Lambert, 1998). They showed that binding of the general anesthetic etomidate onto GABAA receptors requires that the respective receptor harbors a β-subunit (Belelli, Pistis, Peters, & Lambert, 1999). Furthermore, the potency of etomidate to act as a positive modulator at GABAA receptors is abolished by a single amino acid substitution in the β-subunit (Belelli,


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Lambert, Peters, Wafford, & Whiting, 1997; Hill-Venning, Belelli, Peters, & Lambert, 1997). In addition, the finding that different point mutations on GABAA receptors altered the potency of anesthetic agents in a drug-specific manner suggested the existence of multiple, agent-specific binding sites (Belelli et al., 1996). Taken together, these observations argued against a common molecular mechanism of anesthetic action. The observation that the modulatory action of the intravenous anesthetics etomidate and propofol at GABAA receptors require the presence of a β-subunit points to an important principle that also applies to interactions between benzodiazepines and GABAA receptors. Not all subtypes of the GABAA receptor are similarly sensitive to sedative drugs and anesthetic agents. The benzodiazepine binding site is formed by an α1–3,5 and a γ-protein subunit (Rudolph & Knoflach, 2011). Receptors lacking these α-subunits or the γ-subunit are insensitive to benzodiazepines. Similar to these observations, the intravenous anesthetics etomidate and propofol only act on a subgroup of GABAA receptors (Hill-Venning et al., 1997). However, most anesthetics in current use display largely overlapping selectivity profiles, with volatile anesthetics to be the less selective group of compounds (Belelli et al., 1999). Furthermore, it is important to recognize that receptor selectivity is always a matter of concentration. By increasing the concentration of a drug, the number of molecular targets is also increased. This rule applies to general anesthetic agents, even when considering only the relatively narrow range of clinically relevant concentrations. The effects produced by increasing the concentration of an anesthetic can be surprisingly complex. For example, at subanesthetic concentrations, the volatile anesthetic sevoflurane inhibited neurons in the ventral horn of the spinal cord to a large part by enhancing GABAA receptormediated inhibition (Eckle, Hauser, Drexler, Antkowiak, & Grasshoff, 2013). However, after increasing the concentration of sevoflurane, the same agent reduced the same neurons’ excitability predominantly via glycine receptors whereas the impact of GABAA receptors was attenuated. The latter action was caused by an inhibitory action of sevoflurane on GABAergic neurons, thereby reducing GABA-release in the ventral horn of the spinal cord.

4. NEUROANATOMICAL SUBSTRATES FOR GENERAL ANESTHETICS For understanding the role of GABAA receptors in general anesthesia, it is useful to ask how general anesthesia can be defined and how the essential

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components of this state can be measured. Basic components of the anesthetic state that are most consistently addressed in the literature are unconsciousness, immobility and amnesia (Campagna et al., 2003; Hemmings et al., 2005; Rudolph & Antkowiak, 2004). However, the definition of the essential components substantially varies between authors but also changes with time. For example, 50 years ago general anesthetic compounds were primarily defined by their immobilizing properties (Eger, Saidman, & Brandstater, 1965). Nowadays, attention shifted toward the question of how anesthetics cause the loss of consciousness (Alkire, Hudetz, & Tononi, 2008; Franks, 2008). However, a generally accepted definition of general anesthesia is clearly lacking. In my own opinion, unconsciousness, amnesia, and immobility are all indispensible aspects of general anesthesia. About 20 years ago, Kissin launched the hypothesis that different components of the anesthetic state involve drug effects in different parts of the CNS (Kissin, 1993). It is now widely accepted that general anesthetic agents cause immobility predominantly by inhibiting spinal neurons. This hypothesis is built on several lines of evidence. First, transsection of the upper thoracic spinal cord in rats or precollicular decerebration only mildly altered the capacity of volatile anesthetics to ablate painful stimuli-induced movements (Rampil, 1994; Rampil, Mason, & Singh, 1993). In addition, delivery of anesthetic drugs specifically to the brain as opposed to the spinal cord dramatically increased the concentration that was needed to suppress painful stimuli-induced movements (Antognini & Schwarz, 1993). In contrast to immobility, the neuronal substrates involved in producing unconsciousness are less clear. Alkire and colleagues proposed that anesthetics cause functional disconnection in the cerebral cortex, thereby interrupting intracortical communication (Alkire et al., 2008). It is reasoned that the loss of the brain’s capacity to integrate information is causally related to anesthetic-induced loss of consciousness. Loss of function of cortical circuits may involve direct molecular actions of anesthetic drugs on cortical neurons, as indicated by comparing the effects of anesthetic drugs in vivo and in excised tissue slices, containing a part of the neocortical network (Hentschke, Schwarz, & Antkowiak, 2005; Lukatch & MacIver, 1996). Other researchers focused on pathways of sleep and arousal, emphasizing similarities between sleep and anesthesia (Nelson et al., 2002). Taken together, unconsciousness is predominantly produced by anesthetic actions in the brain but the exact neuroanatomical sites that are involved have not been identified without doubt. However, when considering the molecular mechanisms of anesthetic actions, the distinction that anesthetics produce hypnosis and amnesia in the brain whereas immobility is related to drug effects


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in the spinal cord is important because the brain and spinal cord express different populations of anesthetic-sensitive receptors.

5. HOMEOSTATIC REGULATIONS IN KNOCKOUT ANIMALS The effects of general anesthetic agents have been investigated in knockout mice lacking a subunit of the GABAA receptor. Yet, this approach has met with variable success. In several cases, it turned out that a comparison between drug actions in wild-type and knockout animals can be misleading. For example, the sedative action of diazepam was found to be enhanced in α1-knockout mice (Kralic et al., 2002). This observation might suggest that α1-containing GABAA receptors do not contribute to the sedative properties of diazepam. However, this conclusion seems to be wrong as it stands in contrast to other results. For example, zolpidem, a drug that preferably targets α1-containing GABAA receptors produces robust sedation (Crestani, Martin, Mohler, & Rudolph, 2000). Furthermore, studies on α1-knockin mice suggested a dominant role of α1-containing receptors in causing diazepam-induced sedation (Rudolph et al., 1999). One plausible explanation for unexpected results produced by studies on knockout animals is the induction of compensatory physiological regulations. This may result in an altered abundance of other receptors targeted by the drug under consideration (Fritschy & Panzanelli, 2014). It is worth mentioning that these compensations may not be restricted to the GABAergic system. For example, Brickley and coworkers have shown that the loss of GABAA receptors in cerebellar granule cells triggers a form of homeostatic plasticity leading to a profound change in the abundance of two-pore domain TASK-1 channels (Brickley, Revilla, Cull-Candy, Wisden, & Farrant, 2001). This compensation fully maintains the normal physiological function of granule cells. Interestingly, the function of two-pore domain channels is modulated by several volatile general anesthetics (Heurteaux et al., 2004). Thus compensatory regulations give rise to different mechanisms of anesthetic actions in wildtype and knockout mice. Although in experimental studies not always appreciated, the issue of homeostatic regulations is fascinating and the underlying molecular and cellular mechanisms are highly relevant in clinical settings. For example, on the intensive care station, patients may have need for sedation over longer periods of time. The presence of sedative drugs in the CNS is associated with a marked reduction in overall brain activity. On the other hand it is known

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that a long-lasting reduction of neuronal activity induces regulatory mechanisms which try to keep the brain at work. These mechanisms have been investigated in some detail in vitro, shedding light onto the huge capacity of neuronal circuits to adapt to novel conditions (Turrigiano, 1999). In view of these findings, it is very likely that the persistent presence of sedative drugs will affect the expression pattern of GABAA receptors, giving rise to altered requirements in dosing. Similarly, it is very likely that pathological conditions such as inflammation, sepsis, or alcoholism go along with pronounced changes in the GABAergic system. It is an important issue to identify and to understand these feedback-processes in order to establish evidence-based therapies, instead of using trial-and-error approaches in clinical anesthesia, especially with regard to the long-term use of sedative drugs. Research into this issue is required as the results of compensatory regulations cannot be predicted simply by intuition, as exemplified by the observation that α1knockouts are more sensitive to diazepam as compared to wild-type animals (Kralic et al., 2002).

6. ANESTHETIC-RESISTANT MICE For elucidating the role of an ion channel in general anesthesia, it is advisable to check first whether its sensitivity to anesthetic drugs falls within the range of clinically relevant concentrations. However, responsiveness to clinically used concentrations is a necessary, but not a sufficient requirement. Multiple alternative molecular targets for anesthetics may exist in the CNS, displaying a similar or even higher sensitivity. Furthermore, the impact of an alternative target may be higher if it resides on sites allowing highly effective output control. For example, ion channels located on the axon initial segment may have a stronger impact on action potential generation than ion channels sited on distal dendrites. For answering the question whether an ion channel, which responds to clinically relevant concentrations of anesthetic agents, is in fact important for mediating general anesthesia, one possible strategy is to render this channel resistant to anesthetic compounds without affecting its physiological function. Furthermore, it is necessary to generate experimental animals that carry this anesthetic-resistant ion channel, but not the corresponding wild-type channel. If the manipulated ion channel is involved in causing anesthesia, the dose requirement to produce loss of consciousness or immobility in animals carrying the anestheticresistant ion channel should be higher as compared to wild-type animals.


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Thus, for testing the impact of a specific receptor in mediating anesthesia, it is mandatory to compare the actions of anesthetic agents in wild-type and mutant animals, harboring genetically modified receptors. If the drug would take effect only via a single molecular target, compensatory regulations should be of minor importance. However, there is ample evidence in the literature that at clinically relevant concentrations general anesthetics alter the function of multiple ion channels. Thus, the use of animal models may give rise to wrong conclusions if the drug affects ion channels that undergo homeostatic regulation. Therefore, the best approach for assessing the role of a specific ion channel in mediating anesthesia is to render the respective protein insensitive to anesthetics without altering its function. This can be achieved in principle by a point mutation located in or close to the drug’s binding site on the respective receptor. Because, in the best case, the function of the receptor is not affected by the point mutation, regulatory compensations are absent in the mutant animals. One experimental strategy that closely matches these requirements is the knockin transgenic approach (Rudolph & Mohler, 2004). Using this strategy, a mutant gene is inserted at the exact site of the genome where the corresponding wildtype gene is located. This ensures that the effect of the mutant gene is not affected by the activity of the endogenous locus. The intravenous anesthetic etomidate was a highly attractive candidate for testing the relevance of its molecular actions to produce anesthesia because in vitro studies showed that this agent requires the presence of a β-protein subunit for its binding to GABAA receptors (Belelli et al., 1999). Furthermore, there was evidence that the drug’s binding to GABAA receptors is strongly reduced by exchanging a single amino acid in the β-subunit, leaving the function of the receptor intact (Belelli et al., 1997). And finally, in vitro work had also indicated that the potency of etomidate at β2- and β3-subunit-containing receptors is much higher than at β1-containing receptors (Hill-Venning et al., 1997). In view of these findings, it was speculated that the anesthetic properties of etomidate are predominantly mediated by GABAA receptors containing β2- or β3-protein subunits. However, at this point it is important to note that for the reasons discussed above, the relevance of ion channels in general anesthesia cannot be proven by in vitro studies. Furthermore, at clinically relevant concentrations, etomidate not only acts via GABAA receptors. For example, this anesthetic also inhibits the enzyme 1β-hydroxylase, which catalyzes steroid biosynthesis in the adrenal cortex (Engelhardt & Weber, 1994). This action is a serious problem in clinical anesthesia as it is associated with increased mortality in severely ill patients.

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Therefore etomidate is only given as bolus injection and for short duration. In addition, it has been shown that etomidate acts as an agonist at α2Badrenoceptors, thereby increasing arterial blood pressure, an effect that might contribute to the cardiovascular stability of patients after induction of anesthesia with etomidate (Paris et al., 2003). Thus, the exact role of GABAA receptors in etomidate anesthesia remained to be elucidated until experiments on knockin mice were carried out.

7. THE HYPNOTIC ACTION OF ETOMIDATE Two competing groups established mouse models that carried point mutations in the β3- or β2-protein subunit of the GABAA at a position homologous to the mutation in the Drosophila melanogaster Rdl GABAA receptor, which is insensitive to etomidate. In β3(N265M) mice created by Uwe Rudolph’s group in Z€ urich, the amino acid asparagine at position 265 was replaced by a methionine. Immunoblotting and immunohistochemical analysis did not indicate changes in either the expression levels or distribution pattern of the altered β3-subunit or other GABAA receptor subunits ( Jurd et al., 2003). Similarly, characteristics of GABAA receptormediated synaptic transmission in tissue slices derived from the mouse neocortex and spinal cord of wild-type and β3-knockin mice were indistinguishable in the absence of anesthetic drugs (Drexler, Jurd, Rudolph, & Antkowiak, 2009; Grasshoff, Jurd, Rudolph, & Antkowiak, 2007). Standard behavioral tests were used for comparing anesthetic effects of etomidate in wild-type and knockin mice. Because it has been shown that the concentration of many general anesthetic agents causing the loss of righting reflex in rodents well correlates with their concentration causing loss of consciousness in humans, this animal model is routinely used in preclinical drug research for predicting unconsciousness in humans (Franks, 2008). Jurd and coworkers compared the duration of the loss of the righting reflex in wild-type and β3-knockin mice after intravenous bolus injection of 10 mg/kg etomidate ( Jurd et al., 2003). In wild-type mice, the righting reflex was abolished for about 40 min but only for 10 min in β3-knockin mice. In the same study, significant reductions in the duration of the loss of righting reflex were also observed at a lower and a higher dose of etomidate. These findings were interpreted as follows: GABAA receptors containing the β3-protein subunit, which represent a minor GABAA receptor subtype in the brain, substantially contribute to the hypnotic action of etomidate. However, these receptors are not the exclusive targets of


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etomidate in causing loss of consciousness, as the duration of the loss of righting reflex was shortened but not completely abolished. At this point the question emerges, what other receptors may be involved in mediating the hypnotic action of etomidate. A major candidate were GABAA receptors containing β2-subunits because etomidate is almost similarly effective in modulating β2- and β3-subunit-containing receptors. According to the approach taken by Jurd and coworkers, a group at Merck changed an asparagine residue at position 265 of the β2-subunit to serine, the residue that appears at this site in the β1-subunit (Reynolds et al., 2003). Receptors containing this mutation are largely insensitive to etomidate. Intravenous bolus injection of 10 mg/kg etomidate abolished the righting reflex in wild-type mice for about 30 min, but only for about 10 min in β2-knockin mice. This finding supports the hypothesis that β2- and β3containing GABAA receptors almost equally contribute to the hypnotic action of etomidate.

8. ETOMIDATE-INDUCED HYPNOSIS AND SUBTYPESPECIFIC ELECTROENCEPHALOGRAM SIGNATURES Anesthetic actions in the brain correlate with characteristic patterns in the electroencephalogram (EEG; Brown, Lydic, & Schiff, 2010; Rampil, 1998). Very deep anesthesia is paralleled by an almost complete lack of brain electrical activity, whereas so-called burst suppression patterns are observed during deep anesthesia. Small and moderate anesthetic concentrations cause a slowing of oscillatory activity that is associated with an increase in amplitudes. Butovas and coworkers chronically implanted electrode arrays in the prefrontal cortex and hippocampus of wild-type and β3-knockin mice (Butovas, Rudolph, Jurd, Schwarz, & Antkowiak, 2010). Using this experimental approach, it was possible to monitor brain electrical activity before and after intravenous injection of 10 mg/kg etomidate. In the cortex and hippocampus of wild-type mice, injection of etomidate evoked isoelectric baselines and subsequent burst suppression patterns. These actions were strongly attenuated by the β3-knockin mutation. These findings suggest that GABAA receptors containing β3-protein subunits mediate to a large extent etomidate-induced changes in brain electrical activity. The results of similar recordings carried out in β2-knockin mice supported the latter conclusion. Reynolds and coworkers did not find any difference between the burst suppression patterns in wild-type and knockin mice after i.v. injection of 10 or 12.5 mg/kg etomidate (Reynolds et al., 2003). This observation was indeed

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surprising since at the same dose the duration of the loss of righting reflex was significantly shortened in β2-knockin mice.

9. BENZODIAZEPINE-INDUCED SEDATION DOES NOT MANIFEST IN THE EEG It is commonly believed that there is a close relationship between the concentration of an anesthetic in the brain, the degree of suppression of brain functions and anesthetic-induced patterns of brain electrical activity (Alkire et al., 2008). This causal correlation provides the foundation for EEG-based depth of anesthesia monitoring. However, in β2-knockin mice, the duration of the loss of righting reflex was significantly shortened as compared to wild-type animals, but etomidate-induced patterns of brain electrical activity were not different from wild-types, indicating a dissociation between the depth and duration of anesthesia and the EEG signatures (Reynolds et al., 2003). To put this finding into a broader context, it is enlightening to refer to the EEG signatures of benzodiazepines. Tobler and coworkers explored the actions of diazepam in mice carrying point mutations in the α1-, α2-, and α3-protein subunits of GABAA receptors, rendering receptors incorporating the mutated subunits insensitive to diazepam (Tobler, Kopp, Deboer, & Rudolph, 2001). Recordings were performed during the dark phases of the circadian rhythm, when animals were active. Unexpectedly, the characteristic effects of diazepam on the sleeping and waking EEG were indistinguishable in wild-type and α1knockin mice. From this finding, the authors concluded that the motorsedative action of diazepam and its well-known EEG “fingerprint” are not mediated by the same receptors, as only the former effect was absent in α1-knockin mice (Rudolph, Crestani, Tobler, et al., 1999). Studies using subunit-specific antibodies provided evidence that α1-subunits are mostly colocalized with β2- and γ2-subunits in the same GABAA receptors (Olsen & Sieghart, 2009). Furthermore, these receptors form the most abundant GABAA receptor subtype in the brain. Along this logic, it seems that GABAA receptors composed of α1-, β2-, and γ2-subunits cause sedation but do not contribute to the EEG “fingerprint” produced by diazepam. In other words, a clear EEG-correlate for the motor-sedative action of diazepam, probably mediated by α1–β2–γ2-receptors is lacking. Given the difference between the wild-type mice and the β2-knockin mice created by Reynolds and coworkers is mostly defined by α1–β2–γ2 GABAA receptors lacking sensitivity to etomidate in the mutants: What would we expect


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regarding the effect of etomidate in β2-knockin mice as compared to wildtype animals? Because the findings of Tobler and coworkers suggest that positive modulation of putative α1–β2–γ2 receptors does not translate into major changes in the EEG, and because the mechanisms of action of diazepam and etomidate at GABAA receptors seem to be similar on the molecular level, a large difference between the EEG effects of etomidate in wild-type and β2-knockin mice is not expected, although etomidateinduced anesthesia is deeper and lasts longer in wild-types. Thus there is consistency between the disability of diazepam and etomidate to alter EEG patterns via α1–β2–γ2 receptors, although both agents produce sedation (Fig. 1). However, it is important to note, that these findings do not suggest that pharmacological modulation of α1-containing GABAA receptors does not manifest in a change in brain electrical activity. In a recent study, we investigated the effects of diazepam in triple knockin mice, in which only GABAA receptors containing α1-subunits were sensitive to diazepam (Hofmann et al., submitted). It was observed that diazepam

Figure 1 More than 50% of all GABAA receptors in the CNS incorporate an α1- and a β2-subunit. Positive modulation of these receptors by diazepam, etomidate and propofol produce amnesia and sedation. Furthermore, this α1–β2 subtype is involved in causing the loss of consciousness. The sedative properties of benzodiazepines are mediated by GABAA receptors sited on forebrain glutamatergic neurons. Action potential firing of these neurons is significantly reduced by small concentrations of diazepam (10–100 nM) and small concentrations of etomidate (100–200 nM). Thus, this reduction in neuronal activity may be the cause of the sedative effect of benzodiazepines and anesthetic agents. Surprisingly, drug-induced modulation of this major GABAA receptor subtype does not manifest in EEG recordings.

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significantly reduced power density in all frequency bands via modulating α1-containing receptors, an effect that goes along with a prominent depression of action potential activity in the cerebral cortex. However, it seems that in wild-type and single knockin mice this subtle action is difficult to resolve because it is masked by the much stronger effects mediated by other GABAA receptor subtypes.

10. DIFFERENT ROLES OF α2- AND α3-SUBUNITS IN MODULATING BRAIN ELECTRICAL ACTIVITY In two separate studies, Kopp and colleagues explored the contribution of α2- and α3-containing subunits to the EEG “Fingerprint” of diazepam, making use of knockin mice. In α2-knockin mice, most effects of diazepam seen in wild-type controls were absent (Kopp et al., 2004). Contrastingly, the effects of diazepam on the EEG of α3-knockin mutants were indistinguishable from those of wild-type littermates (Kopp, Rudolph, Keist, & Tobler, 2003). The latter result came as a surprise as α3-containing GABAA receptors are heavily expressed in GABAergic neurons of the reticular thalamic nucleus, suggesting that these neurons are not contributing to the effect of benzodiazepines on the EEG. Taken together these findings point to a prominent role of α2-containing GABAA receptors in mediating the actions of benzodiazepines on rhythmic brain activity. In summary, studies on the effects of diazepam and etomidate in α- and β-knockin mice lead to the hypothesis that GABAA receptors harboring α2- and β3-protein subunits affect oscillatory brain activity and mediate to a large extent the well-known actions of benzodiazepines and intravenous anesthetic agents on the EEG (Fig. 2). As already mentioned, the α1–β2–γ2-subunit combination represents the largest population of GABAA receptors in the brain (about 60%), followed by α2–β3–γ2 (15–20%) and α3–βn–γ2 (10–15%) combinations (Rudolph & Knofloch, 2011; Whiting, 2003). Furthermore, it was argued that the latter subtype is unlikely to have a strong impact on brain electrical activity because no difference was found between the action of diazepam in wild-type and α3-knockin mice (Kopp et al., 2003). These observations are promoting the conclusion that α2–β3–γ2-receptors might mediate to a large extent the effects of benzodiazepines and similarly of etomidate on the EEG. Indeed, this hypothesis is compatible with all experimental data available so far. In cortical circuits, α2- and β3-containing receptors are enriched at the axon initial segment of pyramidal cells and at somatic synapses formed


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Figure 2 Less than 20% of GABAA receptors in the CNS harbor an α2- and a β3-subunit. Receptors containing the α2-subunit are mediating the anxiolytic and muscle relaxant properties of benzodiazepines. Furthermore, β3-subunit-containing receptors mediate in part the hypnotic and mostly the immobilizing actions of etomidate and propofol. Drugs acting via this GABAA receptor subtype reduce action potential firing of cortical and spinal neurons, but maximal inhibition via this route is limited, leveling at around 50%. In sharp contrast to the α1–β2 subtype, pharmacological modulation of α2–β3 receptors by benzodiazepines and anesthetic agents translate into changes in brain electrical activity that are evident in the EEG.

between cholecystokinin-positive basket cells and pyramidal cells (Brunig, Scotti, Sidler, & Fritschy, 2002; Freund & Katona, 2007). These anatomical sites allow powerful modification of action potential generation of cortical pyramidal cells.

11. INTRACORTICAL ACTIONS OF ETOMIDATE Angel and Arnott investigated the effect of etomidate on neuronal responses evoked by somatosensory stimuli in the thalamus and cortex of rats (Angel & Arnott, 1999). They observed that etomidate did not alter the excitability of thalamic neurons but caused a concentration-dependent reduction in cortical responsiveness, indicating that cortical networks are highly sensitive to etomidate. Drexler and coworkers tested the latter hypothesis by quantifying the effects of etomidate on electrical activity in cultured tissue slices derived from the neocortex of wild-type and β3-knockin mice (Drexler, Roether, Jurd, Rudolph, & Antkowiak, 2005). At a clinically relevant concentration of 200 nM, etomidate reduced action potential firing of wild-type

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cortical neurons by about 70%, but only by about 30% in tissue slices taken from β3-knockin mice (Drexler et al., 2005). Assuming that etomidate acts via β2- and β3-containing GABAA receptors, this finding suggests that both receptor subpopulations almost equally contribute to the inhibitory action of etomidate. This result well compares to the effectiveness of etomidate to abolish the righting reflex in wild-type and β3-knockin mice ( Jurd et al., 2003). In addition, the authors analyzed the effects of etomidate on rhythmic population activity produced by etomidate by recording local field potentials (LFPs). In slices derived from wild-type mice, etomidate (200 nM) amplified oscillatory population activity in the θ-frequency band (Drexler et al., 2005). Because at the same concentration etomidate profoundly depressed action potential firing of cortical neurons by 70%, the increased θ-band power that was apparent in LFP recordings indicates a synchronizing action of the drug. Interestingly, this synchronizing effect was specifically mediated by β3-containing receptors because it was completely absent in slices derived from β3-knockin animals. It is interesting to note that a boosting effect of etomidate on θ-band oscillations was also observed in vivo (Butovas et al., 2010). In wild-type mice, a strong increase in hippocampal θ-band power transiently emerged during the period the righting reflex recovered and animals regained consciousness. Similar to the in vitro study mentioned above, boosting of θ-band power was only seen in wild-type animals but not in β3-knockin mice. How do these findings compare to the possible roles of α1–β2–γ2- and α2–β3–γ2-GABAA receptors in mediating anesthetic effects on the EEG? The former subtype is efficient in producing motor sedation but has little impact on oscillatory brain activity. Furthermore, motor sedation is mediated by glutamatergic neurons in the forebrain (Zeller et al., 2008). If sedation occurs without marked changes in rhythmic brain activity: What is the physiological basis of the robust sedating action of benzodiazepines? Because GABAA receptors containing α1- and β2-subunits mediate sedation on the behavioral level on the one hand and on the other hand significantly decrease action potential firing of cortical neurons (Drexler et al., 2005), it seems well possible that the decrease in action potential firing, which is indicative of a decrease in neuronal excitability, gives rise to impaired cognitive performance, or, in other words gives rise to sedation. However, GABAA receptors containing the β3-subunit are also involved in decreasing action potential firing. But unlike receptors incorporating the β2-subunit, these receptors seem to play an important role in synchronizing cortical neurons, thereby enhancing oscillatory activity apparent in the EEG


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and in LFP recordings. The ability of isolated cortical networks to largely reproduce anesthetic-induced activity patterns seen in vivo also has been shown in landmark studies conducted by Bruce MacIver and coworkers (Lukatch & MacIver, 1996; MacIver, Mandema, Stanski, & Bland, 1996). These authors demonstrated that the typical activity patterns seen in the EEG under anesthesia can be reproduced in acutely isolated neocortical slices. However, enhancement of oscillatory activity by anesthetics is clearly limited to a range of small and intermediate concentrations. This is because rhythmic activity requires the firing of neurons. At small and intermediate concentrations, anesthetics reduce action potential firing but their strong synchronizing action dominates, resulting into an overall enhancement of rhythmic population activity. This enhancement manifests as an increase in the amplitude of voltage fluctuations seen in the LFP and an increase in the persistence of oscillatory population activity. Within a limited range of anesthetic concentrations, oscillatory activity persists. However, at high anesthetic doses, the inhibitory action of anesthetic agents suppresses synchronized population activity. Isoelectric activity occurs at rather high doses and is characterized by the total lack of neuronal activity (Butovas et al., 2010).

12. ACTIONS OF ETOMIDATE IN THE HIPPOCAMPUS Amnesia is an important goal of general anesthesia. As a rule, recall of intraoperative sensations is unpleasant and frequently triggers posttraumatic stress disorder (Leslie, Chan, Myles, Forbes, & McCulloch, 2010). Remarkably, amnesia is induced by many anesthetic agents at concentrations well below those which cause unconsciousness and immobility (Campagna et al., 2003; Dutton et al., 2001). In fact, in patients and experimental animals the state of conscious amnesia can be achieved purposefully (Veselis, Reinsel, & Feshchenko, 2001). Several brain areas are involved in memory formation, including the hippocampus, amygdala, and prefrontal cortex. Anesthetic actions, possibly related to their memory-impairing effect, have been studied in some detail in the hippocampus (Banks & Pearce, 1999; Lukatch & MacIver, 1996; MacIver & Roth, 1988; Nishikawa & MacIver, 2000). GABAA receptors which contain α5-subunits are highly enriched in hippocampal neurons, where they comprise about 25% of all GABAA receptors (Pirker, Schwarzer, Wieselthaler, Sieghart, & Sperk, 2000). In hippocampal pyramidal neurons, α5-containing receptors are present outside the synapse (Fritschy & Brunig, 2003). These extrasynaptic receptors are activated by small concentrations of GABA that are always

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present in the extracellular space. In addition, α5-containing GABAA receptors sited on the apical dendrites of hippocampal and neocortical pyramidal cells contribute to a form of synaptic inhibition that has been termed GABAA-slow (Banks, Li, & Pearce, 1998; Sceniak & MacIver, 2008; Zarnowska, Keist, Rudolph, & Pearce, 2009). Interstingly, tonic GABAergic inhibition of hippocampal neurons that is mediated by α5subunit containing receptors is enhanced by small concentrations of etomidate and isoflurane that did not alter fast synaptic transmission (Caraiscos et al., 2004; Cheng et al., 2006). Similarly, at small concentrations that did not affect fast somatic inhibition of hippocampal pyramidal cells, etomidate increased the amplitude of GABAA-slow (Dai, Perouansky, & Pearce, 2009). Taken together, these findings implicate a role of α5subunit-containing GABAA receptors in conscious amnesia. The latter conclusion is also backed by studies on α5-null mutant mice. While etomidate impaired spatial and nonspatial hippocampal-dependent learning tasks, this was not observed in null mutant mice (Cheng et al., 2006). Interestingly, sedative and hypnotic effects of etomidate were not different in wild-type and α5-null mutant mice, suggesting that α5-subunit-containing GABAA receptors do not contribute to these anesthetic endpoints. But how does modulation of GABAA receptors harboring α5-subunits translate into memory impairment? In one scenario, increased inhibition of hippocampal pyramidal cells may prevent entry of calcium ions through N-methyl-D-aspartic acid (NMDA) receptors, and the ensuing cascades that result in long-term potentiation (Simon, Hapfelmeier, Kochs, Zieglgansberger, & Rammes, 2001). The kinetic characteristics of GABAA-slow as well as its presence in the dendrites of pyramidal cells strongly suggests a central role in controlling NMDA receptor-dependent plasticity. However, in another scenario, anesthetics may disturb the highly organized rhythmic activity patterns that are thought to be essential for hippocampal learning (Perouansky & Pearce, 2011). For example, a change in hippocampal θ-oscillations might be sufficient to interfere with plastic changes underlying memory formation. Can we conclude from these observations that in patients anesthetized with etomidate, isoflurane, or propofol, amnesia is mediated exclusively or predominantly by GABAA receptors containing α5-subunits? Probably not, because these agents preferably act via α5-containing receptors only if small drug concentrations are administered that are not sufficient to produce unconsciousness. At higher, clinically relevant concentrations, further subtypes of GABAA receptors are modulated by anesthetic agents. Studies on knockin and knockout mice provided compelling evidence that


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α1-containing receptors are contributing to the amnestic properties of benzodiazepines (Rudolph, Crestani, Benke, et al., 1999) and of inhaled anesthetics (Sonner et al., 2005). Because α1-containing receptors are the prevalent subtype in the forebrain, it is very likely that in clinical anesthesia these receptors contribute in a prominent manner to the impairment of memory formation produced by anesthetic agents.

13. SPINAL ACTIONS OF ETOMIDATE The hindlimb withdrawal reflex is commonly used as a measure of the immobilizing activity of drugs. In β2-knockin mice, the duration of the loss of the hindlimb withdrawal reflex after intravenous injection of 10 and 15 mg/kg etomidate was somewhat shorter than in wild-type animals (Reynolds et al., 2003). However, at the same concentrations, etomidate completely failed to cause the loss of the hindlimb withdrawal reflex in β3-knockin mice ( Jurd et al., 2003). These findings indicate β3-subunit-containing GABAA receptors as the most important, although not exclusive molecular pathway by which etomidate ablates painful stimuli-evoked protective reflexes (Fig. 2). The ventral horn of the spinal cord is a key structure for anesthetic agents to produce immobility ( Jinks, Bravo, & Hayes, 2008; Kungys, Kim, Jinks, Atherley, & Antognini, 2009). Therefore, clinically relevant concentrations of etomidate were expected to reduce the activity of ventral horn neurons in wild-type animals. Moreover, the finding that etomidate failed to induce the loss of the hindlimb withdrawal reflex in β3-knockin mice predicted that etomidate-induced inhibition of ventral horn neurons should be mediated to a large extent by GABAA receptors containing the β3-subunit. To test the latter hypothesis in vitro, actions of etomidate were compared in tissue slices that were prepared from the spinal cord of wild-type and β3-knockin mice. Electrophysiological recordings were conducted for quantifying the concentration-dependent effects of the anesthetic on the discharge rate of ventral horn neurons. Consistent with the ideas lined out above, etomidate inhibited action firing of neurons in spinal slices taken from β3-mutant mice to a much lower extent as compared to wild-type neurons (Grasshoff et al., 2007). Unexpectedly, etomidate failed to completely depress action potential firing of wild-type neurons even when applied at very high concentrations. For etomidate, but also for the intravenous anesthetic propofol, concentration response curves leveled at a maximum of about 60%

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inhibition. This upper limit was reached well within a range of clinically relevant concentrations (around 1 μM). Because studies on expressed GABAA receptors contrasted the hypothesis that the potentiating action of etomidate saturates at this concentration (Belelli et al., 2003), the question was further elucidated how the molecular effects of etomidate on GABAA receptors translate into changes in action potential firing. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were recorded from voltage clamped ventral horn neurons derived from wild-type mice. As expected, etomidate concentration-dependently prolonged the current decay time of GABAergic IPSCs (Grasshoff et al., 2007). In contrast to the effect of etomidate on action potential firing, this effect did not saturate at a concentration of 1 μM. In neurons derived from β3-knockin mice, etomidate did not significantly alter current decay times of GABAergic IPSCs, indicating that its potentiating action in wild-type neurons was mostly mediated by β3subunit-containing GABAA receptors. However, besides changing the decay time of IPSCs, etomidate altered the frequency of synaptic events. At clinically relevant concentrations (around 1 μM), etomidate reduced the rate of occurrence of action potential-dependent GABAergic synaptic events in a concentration-dependent manner. Thus, on the one hand, etomidate potentiated GABAA receptor-mediated IPSCs, but on the other hand this anesthetic inhibited the occurrence of IPSCs, an effect equivalent to neuronal disinhibition. Because the action of etomidate on the decay time of IPSCs and on their frequency of occurrence altered overall GABAA receptor-mediated inhibition of ventral horn neurons in opposing directions, these qualitative different actions on GABAergic synaptic transmission compensate each other, thereby limiting the capacity of etomdiate to depress neuronal excitability. Moreover, in slices prepared from β3-knockin mice, the frequency of action potential-dependent synaptic events was not altered by etomidate. This observation indicates that the release of GABA onto ventral horn neurons is under the control of GABAA receptors harboring β3subunits. Taken together, our studies on the actions of etomidate in the spinal cord provided evidence that enhancing GABAA receptor-function diminishes GABA-release onto ventral horn neurons. Therefore the capacity of agents to reduce the excitability of ventral horn neurons is limited if these agents mostly act via GABAA receptors. Thus, in the ventral horn of the spinal cord a push–pull situation results if anesthetics are administered that potentiate GABAA receptor-function: The inhibitory current flowing in the course of single synaptic events is increased but the frequency of these events is


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reduced. It is interesting to note that in this situation the exact molecular mechanism of action can make a big difference. When investigating the actions of thiopental, a barbiturate that is still used in clinical anesthesia, we observed that this agent prolonged the decay times of GABAA receptor-mediated IPSCs and, at the same time, reduced the frequency of IPSCs (Grasshoff, Netzhammer, Schweizer, Antkowiak, & Hentschke, 2008). These observations exactly correspond to the push–pull mechanism described for etomidate. However, besides changing synaptically mediated GABAergic inhibition, a strong inhibitory tonic current was also induced by the barbiturate that was independent on GABAergic synaptic transmission. Such a current could result from the direct activation of GABAA receptors and therefore does not require ongoing synaptic transmission. Alternatively it could result from a modulating action of thiopental on extrasynaptic receptors. Whatever the case may be. As compared to etomidate, thiopental more profoundly inhibited action potential firing of ventral horn neurons, with a maximal effect leveling at about 90% inhibition. The above observations are prompting the question of how the effects of intravenous anesthetics characterized in vitro relate to clinical anesthesia. The GABAergic anesthetics etomidate and propofol are potent hypnotics but clinical studies have provided ample evidence that their immobilizing capacity is clearly limited (Ashworth & Smith, 1998; Smith & Thwaites, 1999; Watson & Shah, 2000). This limited capacity is explained by an inhibitory action on GABAergic neurons, thereby reducing GABA-release in the ventral horn of the spinal cord.

14. ANESTHETIC SIDE EFFECTS The type of anesthetic procedure and the choice of anesthetic agents depends on several factors including the physical state of the patient and the kind of surgery to be performed. Today, general anesthesia is very safe for young and healthy patients. However, the proportion of old people to undergo surgery and to require general anesthesia is steadily increasing. In these patients, side effects of anesthetic agents may become a serious safety factor. However, our knowledge on the molecular mechanisms that underlie the unwanted and dangerous side effects of general anesthetics is very limited. Anesthetic agents produce hypothermia and postoperative shivering. This effect is observed in about 50% of the patients, when using volatile anesthetics and in about 15% of patients after propofol anesthesia (De Witte & Sessler, 2002). Unfortunately, the reduction of core body

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temperature causes adverse cardiac events, increases bleeding and impairs wound healing (Frank et al., 1997; Kurz, Sessler, & Lenhardt, 1996). The main control centers of body temperature are the preoptic area and the anterior hypothalamus. Studies on knockin mice lead to the conclusion that β2containing GABAA receptors mediate the hypothermic action of etomidate (Cirone et al., 2004). Another well-known side effect of general anesthetics is respiratory depression, which occurs with volatile and intravenous anesthetics. This side effect seems to involve GABAA receptors in inspiratory premotor neurons in the caudal ventral respiratory group (Stucke et al., 2005). Investigations on β3-knockin mice suggest that etomidate and propofol cause respiratory depression via ß3-subunit-containing GABAA receptors (Zeller, Arras, Lazaris, Jurd, & Rudolph, 2005).

15. MULTISITE AND MULTIPLE MOLECULAR ACTIONS OF GENERAL ANESTHETICS Our knowledge on GABAA receptor-mediated inhibition rapidly developed in the past decade and its complexity is amazing. GABAA receptors assemble from different protein subunits. The specific subunit composition determines their kinetic features, their sensitivity to agonists, their presence at synaptic or extrasynaptic sites, and their pharmacological properties. The fact that structurally different GABAA receptors show different expression patterns in the CNS underscores their specific physiological functions. By using an in vivo point mutation strategy, it was possible to assign the sedative, hypnotic, and immobilizing properties of intravenous anesthetics to specific subtypes of the GABAA receptor. Furthermore, it was possible to identify neuroanatomical substrates that mediate important components of general anesthesia. It is interesting to compare Hanns M€ ohler’s new benzodiazepine pharmacology and the “multisite and multiple-mechanisms” concept of general anesthesia (Grasshoff, Rudolph, & Antkowiak, 2005; M€ ohler, Fritschy, & Rudolph, 2002). Obviously, there is a large degree of overlap, as both concepts assign different drug effects that manifest on the behavioral level to different molecular targets. Because these targets show specific expression patterns in the CNS, behaviorally distinct drug actions are mediated by different neuroanatomical substrates. However, it is important to note that general anesthetics define a group of compounds displaying great diversity and the number of ion channels that are affected by general anesthetic agents is by far larger than the number of benzodiazepine-sensitive GABAA receptor subtypes. As a consequence,


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the “multisite and multiple-mechanisms” concept appears to be agent specific (Grasshoff et al., 2006). For example, it is likely that the volatile anesthetic sevoflurane and the intravenous anesthetic etomidate both mediate immobility to a large part via acting in the ventral horn of the spinal cord. But the spectrum of molecular targets that is affected by these different compounds seems to differ considerably (Grasshoff et al., 2006). Agent selective and pathway specific actions of general anesthetic agents have been reported by Bruce MacIver and Sheldon Roth a long time ago (MacIver & Roth, 1987a,1987b). When considering the neuronal substrates that are involved in causing unconsciousness, things get even more complex. The great diversity of results provided by imaging studies raises the possibility that different anesthetic agents cause the loss of consciousness via acting in different parts of the brain (Alkire, Haier, Shah, & Anderson, 1997; Alkire et al., 2008; Fiset et al., 1999). Given that the loss of consciousness induced by general anesthetic agents is causally related to the brain’s reduced capacity to integrate information, it seems plausible that there are many different ways to compromise this ability, including direct drug actions on cortical neurons and indirect action on pathways of sleep and arousal.

16. AGENT-SPECIFIC ACTIONS OF ANESTHETICS LACKING BINDING SELECTIVITY In vivo point mutation strategies were successfully used for assigning the sedative, amnestic, anxiolytic, and muscle relaxant properties of nonselective benzodiazepines to specific subtypes of the GABAA receptor (Fig. 2). These exciting findings raised great expectations. Hanns M€ ohler and colleagues wrote: “Rational drug targeting to specific receptor subtypes has now become possible. Only restricted neuronal networks will be modulated by the new subtype-selective drugs” (M€ ohler et al., 2002). The authors reasoned that more selective compounds will provide a novel class of therapeutics, displaying less severe side effects as the nonselective benzodiazepines in current use. In this context, the authors suggested the development of novel drugs acting via a decreased number of molecular targets as compared to the so-called nonselective benzodiazepines. But does this mean that drugs that target multiple receptors in the CNS are nonspecific in a sense that their molecular and systemic actions cannot be distinguished from each other? This question can be addressed by comparing the actions of propofol and etomidate. Studies on expressed receptors showed that these anesthetic agents act via largely overlapping molecular targets in the CNS (Belelli et al.,

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1999). On a first view, these similar molecular actions might suggest that the drugs’ effects on a higher level of complexity are also similar. This hypothesis is based on the existence of a strong causal relation between the molecular and systemic level of drug action. But does this principle of a strong causality, i.e., similar causes result in similar effects, really apply? In fact, dissimilar and even contrasting actions of etomidate and propofol have been reported in clinical anesthesia. For example, both agents were used in electroconvulsive therapy in patients suffering from depression (Avramov, Husain, & White, 1995; Gazdag, Kocsis, Tolna, & Ivanyi, 2004). Interestingly, etomidate and propofol showed opposing actions on the length of seizures. In another study, etomidate, but not propofol enhanced the amplitude of somatosensory evoked potentials (Banoub, Tetzlaff, & Schubert, 2003; Sloan, 1998). In cortical tissue slices, etomidate and propofol differently affected population activity (Drexler et al., 2009). These findings show that although interacting with multiple, largely overlapping molecular targets, the action profiles of etomidate and propofol in patients and in excised tissue slices are clearly different. Thus subtle differences in the spectrum of molecular actions translate into drug-specific actions that manifest on higher levels of neuronal activity. In other words, drugs that lack binding selectivity on the molecular level frequently display individual, fingerprint-like actions, visible in EEG recordings, in clinical anesthesia, and in excised tissue slices. These observations are raising the question as to whether the principle of a strong causality really applies to general anesthetic agents. This is an interesting issue as current drug research and development is largely based on the assumed existence of strong causal relations between the molecular and systemic level.

17. CONCLUSION In clinical anesthesia, a number of different anesthetic compounds are around. These include the volatile anesthetics isoflurane, sevoflurane, and xenon. Intravenously applied agents are propofol, thiopental, etomidate, midazolam, and ketamine. All these drugs are characterized by individual clinical profiles and they are often administered for specific medical purposes, underscoring the need for specific forms of anesthesia in specific clinical settings and in different patients. It seems highly unlikely that in the future all needs of anesthesiologists can be satisfied by a single drug. Indeed, most of the currently used anesthetic agents have been introduced decades ago. Their


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continuous use suggests that these agents cannot be easily replaced by others. However, because of dangerous side effects, the large quantity of people undergoing anesthesia and a steadily increasing proportion of elderly people, there is need for improved drugs. In order to define important features of improved anesthetic agents, a better understanding is required by what combination of molecular targets the essential components of the anesthetic state can be achieved. In particular, we need to know what molecular pathways can be used to induce the mandatory anesthetic actions. It is also important to study unwanted side effects in greater detail in order to figure out how they can be prevented. Furthermore, research into specific requirements of different patients is needed. It is unlikely that newborn children, elderly people, or patients suffering from sepsis are expressing the same set of anesthetic-sensitive receptors and are similarly vulnerable to side effects. The most serious problems in using these anesthetic agents are in fact caused by unwanted side effects. Nonetheless, the currently available anesthetic agents provide a good starting point. Understanding their clinical profiles and the related molecular targets should facilitate the development of structurally related compounds or of agents that combine some of the desired actions of different anesthetics in current use while avoiding dangerous side effects. This approach would be based on our extensive clinical experience with available agents. It has been pointed out that drug development is inherently risky, and a stronger role of science has been recommended to reduce failure rates in drug development (Paul et al., 2010). Interestingly, the concept of designing maximally selective ligands to act on specific drug targets has been questioned in general ( Jia et al., 2009; Pujol, Mosca, Farres, & Aloy, 2010; Russell & Aloy, 2008). The criticism underscores the robustness of biological systems, which is built on redundant pathways, strong feedback connections, and compensatory signaling routes. Thus, modulation of a single molecular target may be insufficient to suppress brain activity triggered by strong sensory stimuli and painfulstimuli evoked reflex pathways mediating emotional, motor, cardiovascular, and immune responses. Network pharmacology may provide an alternative approach. In this concept, signal pathways are considered as networks of molecules and interacting reaction pathways. It is self-evident that this approach calls for a greater role of complex in vitro models and systems biology in drug research and development. In this context, the use of knockin animals and point mutated drug receptors could take center stage. Thus, the combination of genetic approaches, and the use of in vitro test systems that cover complex network structures may help to increase the success rate in preclinical drug development.

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CONFLICT OF INTEREST The author indicates no conflicts of interest.

ACKNOWLEDGMENTS This chapter is dedicated to Professor Bernd W. Urban who is going to retire soon. I would like to thank him for long-lasting support and inspiration coming from his work on anesthetic mechanisms.

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