Mechanisms of general anesthesia: from molecules to mind

Best Practice & Research Clinical Anaesthesiology Vol. 19, No. 3, pp. 349–364, 2005 doi:10.1016/j.bpa.2005.01.004 available online at http://www.sciencedirect.com

3 Mechanisms of general anesthesia: from molecules to mind George A. Mashour1 MD, PhD Clinical Fellow in Anaesthesia, Harvard Medical School, Boston, MA, USA; Resident in Anesthesia and Critical Care Massachusetts General Hospital, 55 Fruit Street/CLN 309, Boston, MA 02114, USA

Stuart A. Forman2 MD, PhD Associate Professor of Anaesthesia, Harvard Medical School, Boston, MA, USA; Associate Anesthetist, Department of Anesthesia and Critical Care Massachusetts General Hospital, 55 Fruit Street/Edwards 5th Floor, Boston, MA 02114, USA

Jason A. Campagna*

MD, PhD

Assistant Professor, Department of Anesthesia and Mahoney Institute for Neurologic Studies, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Longnecker Anesthesia Research Labs, 3612 Hamilton Walk/305 John Morgan Building, Philadelphia, PA 19104-6112, USA

Despite the widespread presence of clinical anesthesiology in medical practice, the mechanism by which diverse inhalational agents result in the state of general anesthesia remains unknown. Over recent decades, our understanding of general anesthetic mechanisms has evolved dramatically from early unitary hypotheses, largely due to the development and influence of a myriad of scientific disciplines ranging from molecular biology to cognitive neuroscience. These discoveries have led to a renaissance of investigation into the mechanisms of general anesthetics and have generated both novel answers and questions. In this chapter, we review the major hypotheses of general anesthetic mechanisms of action and present an expanded overview of current investigation into those mechanisms. We also present a framework to aid in thinking about the actions of these agents, highlighting

* Corresponding author. Tel.: C1 215 662 3759. E-mail addresses: [email protected] (G.A. Mashour), [email protected] (S.A. Forman), [email protected] (J.A. Campagna). 1 Tel.: C1 617 276 3030. 2 Tel.: C1 617 724 5156.

1521-6896/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved.

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the relationship between putative targets at the molecular level and the more integrated functional changes in behavior and consciousness. Key words: general anesthesia; general anesthetics; mechanism of anesthesia; theories of anesthesia.

THEORIES OF GENERAL ANESTHETIC MECHANISMS OF ACTION Anesthesia was first demonstrated successfully in 1846 and within a 10-year period three structurally diverse inhalational anesthetics—chloroform, ether and nitrous oxide—were in widespread use. That there were three, rather than merely one such agent that produced general anesthesia is quite significant. This phenomenon led to the formulation of the fundamental scientific question in anesthesiology: how do volatile gases with diverse structures give rise to the state of anesthesia? Claude Bernard suggested that different anesthetic agents acted through a common final mechanism— and thus was born the original ‘unitary hypothesis’ of general anesthesia. Implicit in Bernard’s framing of his hypothesis is that all anesthetics produce a common unique state. In fact, three major anesthetic endpoints are the foci of most behavioral studies in humans and animals. In the era before muscle relaxants, ablation of motor responses to pain (immobility) was synonymous with ‘anesthesia’. Minimal Alveolar Concentration (MAC) is the potency scale for this anesthetic action. More recently, research has also focused on the hyponotic and amnestic activities of general anesthetics. Hypnosis is more precisely defined as the lack of ‘perceptive awareness’, which is assessed from responses to non-noxious stimuli. The amnestic actions of general anesthetics are among the most potent, and can be assessed both for explicit (conscious) and implicit (unconscious) memory formation. In the late 19th century, Meyer and Overton independently observed that the potency of volatile anesthetics for ablating responses to noxious stimuli was correlated with their solubility in olive oil. The synthesis of these ideas led to the ‘lipid hypothesis’ of general anesthesia, the proposition that anesthetics act by non-specifically perturbing lipid cell membranes of neural tissues.1 This hypothesis provided a foundation for scientific investigation and led to a number of key insights into how general anesthetics act to produce their effects. The lipid hypotheses of general anesthesia persisted late into the 20th century, until being challenged by another unitary construct, the ‘protein hypothesis’. Although there were observations that were long understood not fit well with the lipid hypotheses, the idea that proteins could be directly involved in anesthetic actions did not gain traction until the late 1970s and early 1980s.2–5 Ultimately, the hypothesis that proteins could be the specific and direct targets of inhaled anesthetic gases was made plausible by the work of Franks and Lieb, who showed that a wide variety of anesthetics inhibit a lipid-free preparations of the enzyme firefly luciferase in parallel with their hydrophobicity. Critically, these studies demonstrated that lipid was not required for the actions of volatile agents.6 By the mid 1990s, a dramatic shift in the paradigm guiding researchers had taken place, with the role of proteins as direct targets for anesthetics being the cardinal feature. It has since been shown that volatile anesthetics exert actions on a variety of proteins, from serum proteins such as albumin to ion channels located in the cell membrane to intracellular signaling molecules such as protein kinase C.7 Despite the plethora of documented anesthetic binding proteins, those considered the most likely molecular targets of anesthetics are ion channels.7,8 Ion channels

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establish resting neuronal membrane potentials, propogate action potentials, moderate pre-synaptic neurotransmitter release, and activate in response to post-synaptic neurotransmitter binding. Thus, ion channels are the molecular mediators of dynamic neural activity, which is the basis of behavior and consciousness. The relationship between such molecular events situated in a neuroanatomic context and higher-order cognitive functions is the new horizon for the investigation of volatile anesthetics and is intimately linked to the renaissance of interest in the how these agents act, as well as to the question of the neuroscientific basis of consciousness. In order to survey this horizon, we will discuss the actions of anesthetics at the molecular, anatomic, and behavioral level. Molecular targets of volatile anesthetics Understanding the relationship between actions at the molecular level and the behavioral effects of volatile anesthetics is the ultimate goal of molecular studies of anesthesia. A number of molecular and pharmacologic tools have developed to aid in this task. One useful group of compounds is the so-called ‘non-immobilizers’. These are volatile halogenated alkanes with structural similarities to volatile anesthetics. While predicted by the Meyer-Overton lipid solubility correlation to be potent anesthetics, non-immobilizers lack the ability to inhibit motor responses to pain.9 The focused and insightful application of these compounds to questions about the nature of anesthetic mechanisms has resulted in a number of fundamental observations about anesthesia. Non-immobilizers fail to produce immobility, but like their halogenated anesthetic cousins, do produce amnesia.10,11 Comparing the actions of non-immobilizers to standard anesthetics on specific ion channels has helped to produce a clearer understanding of which channels, among the myriad possible targets, represent those likely involved in the production of general anesthetic actions.7,12 Anesthetic sites on proteins Eckenhoff and Johansson have articulated a general concept of ‘Promiscuous Protein Sites’ that describes a multiplicity of possible anesthetic binding sites in virtually all proteins.13,14 A central feature of this hypothesis is that anesthetic binding is generally weak and non-specific because of the small size of these ligands. Another formulation of this idea of weak binding to protein sites suggests that anesthetics perturb weak hydrogen forces or van der Waals associations of protein folding, or protein–protein interactions so as to affect the functional state of target proteins.15,16 Such weak and non-specific nature of anesthetic binding to proteins makes it difficult to use traditional ligand-binding techniques to locate putative sites. Despite such theoretical difficulties, binding sites for general anesthetics have been identified on a number of ion channels, including the nicotinic acetylcholine and GABAA receptors.17–22 The sites have been identified using a number of recently developed techniques. One approach, ‘reverse pharmacology’, entails the site-directed mutagenesis of selected amino acids or protein segments, followed by testing for alteration in sensitivity to anesthetics. This potentially powerful technique has identified potential anesthetic binding sites on some channels while failing in other putative targets. Another approach to identifying protein sites is to use photo-reactive anesthetic labels, where exposure to specific wavelengths of light catalyzes covalent binding between the labeled compound and the protein site.21,23 Although also a powerful technique, its use is limited by the difficulty of obtaining sufficient quantities of purified protein for study.

352 G. A. Mashour et al Table 1. Roles of some anesthetic-sensitive ion channels in cellular excitability, behavior, physiology, and pharmacology. Ligand-gated ion channels

Cellular role(s)

Gamma-aminobutyric acid type A receptorsa

Increased ClK permeability, membrane hyperpolarization Inhibit excitability

Glycine receptors

Increased ClK permeability, membrane hyperpolarization Inhibit excitability

Neuronal nicotinic acetylcholine receptors

High permeability to monovalent cations and CaCC Modulate release of neurotransmitters

Muscle nicotinic acetylcholine receptors Serotonin receptors

Neuromuscular transmission Enhances excitability via inhibition of resting KC leak currents Fast excitatory neurotransmission Cation conductance for CaCC, MgCC inhibition Cation conductance for CaCC, MgCC inhibition

Glutamate receptorsb NMDA subtype AMPA/Kainate subtype Other ion channels Potassium channels Non-voltage gated background channels e.g. TREK/ TASK Voltage-activated

Cellular role(s)

Modulation of cell resting potential and excitability Chemical, mechanical and pH-sensitive Recovery phase from action potentials

Non-voltage dependent neurotransmitter/ATP activated

Inward rectifying, pH sensitive

Sodium channels

Action potential generation and propagation

Behavioral/physiological and pharmacological role(s) Enhanced activity associated with anxiolysis, sedation, amnesia, myorelaxation, anticonvulsants. Spinal reflexes and startle responses. GlyR is a major inhibitory receptor in spinal cord. Association with memory, nociception, mutations linked with seizure disorders. Autonomic functions. Skeletal muscle contraction Arousal, possible role in emesis

Perception, learning and memory, nociception Perception and memory Behavioral/physiological and pharmacological role(s)) Non-specific; likely widespread

Nerve conduction, cardiac action potentials, mutations associated with cardiac arrhythmias Glucose sensor in b-cells Possible role in ischemic preconditioning Nerve conduction. Cardiac action potentials (arrhythmias)

c

Calcium channels : Voltage-gated cardiac Voltage-gated neuronal Calcium induced calcium release (CICR) Ryanodine receptor Inositol-1,4,5-triphosphate (IP3) receptors

a b c

Pacemaker potentials in neurons (T-type) Presynaptic localization Neurotransmitter release

Cardiac inotropy and chronotropy, vascular tone. Non-specific; likely widespread.

Intra-cellular channels Release of intracellular CaCC stores after stimulation of surface receptors Produces calcium oscillations

Excitation contraction coupling

Excitatory amino acid. Excluding metabotropic receptors. T, N, L, P subtypes.

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Effects of anesthetics on ion channels A variety of ion channels that modulate the electrical activity of cells are linked to the behavioral or physiological actions of anesthetics (Table 1). Many ion channels are sensitive to volatile anesthetics at clinically effective concentrations (Table 2) including voltage-gated channels, the ligand-gated superfamily of ‘cysteine-loop’ neurotransmitter receptors (nicotinic acetylcholine, serotonin type 3, GABAA, and glycine receptors), and the glutamate receptors activated by N-methyl-D-aspartate (NMDA) or (alpha)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA).7,8 We will highlight the effects of inhaled anesthetics on five channel families, two ligand-gated and three voltage-gated, so that a general idea of the nature of the effects of these agents on channels are appreciated. More importantly, a common foundation will be established for exploring how these actions may affect higher, integrative levels, to alter behavior and consciousness, the ultimate endpoints of the anesthetized state. Voltage-gated ion channels. Volatile anesthetics can act to perturb neuronal function via pre-synaptic or post-synaptic mechanisms, or both.24–26 Although the majority of evidence suggests that modulation of post-synaptic function is likely of more importance, there is a growing body of literature that suggests pre-synaptic actions of anesthetics significantly contribute to the production of the general anesthesia.27 Voltage-gated calcium channels that regulate pre-synaptic transmitter release are likely candidate targets.12,28 It has been known for some time that volatile anesthetics affect voltage-gated sodium and potassium channel function on peripheral nerves, albeit at higher concentrations that those used clinically.29,30 In recent years, however, new observations and improved techniques have combined to suggest that related channels in the central nervous system are in fact clinically relevant to anesthetic actions.31–33 The repertoire of likely ion channel targets also includes tandem-two pore or Table 2. Actions of different classes of inhaled anesthetics on ion channels. Functional effect of inhaled anesthetics Ion channel Gamma-aminobutyric acid type A (GABAA) Glycine receptors Neuronal nicotinic acetylcholine receptors Muscle nicotinic acetylcholine receptors Serotonin receptors Glutamate-NMDA Glutamate-AMPA/Kainate Background KC channels (TREK/TASK) Voltage-activated KC channels ATP-activated KC channels Voltage-activated NaC channels Voltage-activated CaCC channels Ryanodine-activated CaCC channels

Halogenated alkanes/ethers

Non-halogenated alkanes

Xenon and nitrous oxide

[

0

0

[ YY

[[ YY

0 Y

Y Weak Y Y Y [[ Y or 0 [ or 0 (agent specific) weak Y weak Y Y or [

Y ND Y ND [ ND ND

ND 0 Y 0 [ 0 ND

weak Y ND ND

ND 0 ND

354 G. A. Mashour et al

‘background leak’ potassium channels. These channels are responsible for maintaining resting membrane voltages in neurons, and volatile anesthetics significantly enhance their activation, thus suppressing responses to excitatory inputs.34 Ligand-gated ion channels. In recent years, GABAA receptors have been the focus of much attention as a possible target for both intravenous and inhaled anesthetics. At clinically effective concentrations, a broad variety of general anesthetics increase apparent GABA sensitivity and prolong inhibitory post-synaptic current (IPSCs) mediated by GABAA receptors.35 These effects augment the normal GABAA-receptormediated inhibition of post-synaptic neuronal excitability.36 Paralleling the enhanced responses of GABAA receptors in vitro, positron-emission tomography in humans demonstrates concentration-dependent anesthetic modulation of GABAA receptors in the brain.37 The potency with which volatile anesthetics enhance the function of GABAA receptors in vitro also parallels in vivo MAC-immobility.38 Taken together, these observations tend to support a central role for GABAA receptors in anesthetic pharmacology and even suggest a common mechanism for many inhaled general anesthetics. The modulation of GABAA receptors, however, is neither necessary nor sufficient to account for every effect of all general anesthetics. The gaseous anesthetics xenon and nitrous oxide only minimally enhance GABA-mediated currents in vitro39–41, and even high concentrations of cyclopropane and butane fail to alter the function of GABAA receptors.42 The above observations, coupled with the findings that clinical concentrations of gases that fail to act on GABAA receptors nonetheless inhibit NMDA-sensitive glutamate channels and neuronal nicotinic acetylcholine receptors, suggest that excitatory ligand-gated ion channels contribute to an alternative pathway to anesthesia. For example, in spinal motor neurons (the ultimate site of anestheticinduced immobility), volatile anesthetics augment the activity of inhibitory glycine receptors and inhibit post-synaptic AMPA and NMDA receptors, in addition to action at GABAA receptors.43 Importantly, the diversity of agents that modulate the glycine receptor is nearly as inclusive as that which acts on the GABAA receptor system.7 The neuronal nicotinic acetylcholine receptors are inhibited by inhaled anesthetics at low concentrations that cause amnesia but not immobility.44,45 Inhaled anesthetics also alter the function of serotonin type 3 (5HT-3) receptors. Some anesthetics potentiate activation of these channels, while other agents show overall inhibitory actions.46 Revisiting the role of lipids Despite the growing focus on protein sites where anesthetics bind and act, a role for lipids in the actions of volatile anesthetics cannot yet be excluded. Many anesthetic target proteins function within lipid membranes. Therefore, it is difficult to discern whether ion channel modulation by anesthetics is caused indirectly by changes in membrane structure or directly by binding to protein sites. There still remain a number of observations that are not explained by a protein-only model of anesthetic actions. For example, the strong cholesterol dependence of nicotinic acetylcholine receptor agonist binding and gating, clearly shows that the channel micro-environment can modulate protein function.2,47 Additionally, alterations in cellular lipid content impacts the potency of inhaled anesthetics.48 In arguing for a direct effects of lipid in anesthetic actions, many ion channels show differential kinetics of opening, closing or inactivation depending upon their lipid micro-environments.49 However, cellular cholesterol depletion also disrupts transport and expression of the AchR, suggesting a more

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indirect role for lipids in mediating anesthetic actions.50 The strong lipid-dependence of channel function and expression constrains the interpretation of in vitro channel studies performed in cells of varying membrane cholesterol and lipid composition to any actual in vivo effects of anesthetics. The ‘lateral pressure hypothesis’ which states that general anesthesia results from the disturbance of lipid membrane biophysical profiles, can explain many of the correlative observations about anesthetics and lipid (Meyer-Overton) as well as mechanistic explanations of anesthetic actions on proteins.51 Experimental paradigms for testing such hypotheses are possible, but are not as accessible as those for protein hypotheses so the idea that volatile anesthetics act to modulate the lipid microenvironment and therefore protein function, remains a viable, though untested general hypothesis. Importantly, the duality or mutual exclusivity of the protein and lipid hypotheses is artificial. The complex interaction of anesthetics with both these biomolecular targets is one of the many unresolved questions contributing to the renaissance of interest in mechanisms of general anesthesia. Genetic studies of anesthetic actions Genetic manipulation of animals is a powerful and increasingly utilized technique for investigating links between potential targets of anesthetics and the behavioral effects of these agents. By screening for mutations in selectively bred populations and by the creation of mutants at putative target sites, an abundance of data that support the hypothesis of anesthetic actions on proteins as being causally related to anesthesia have been generated. Genetic screens of non-mammalian animals have yielded a number of possible targets of anesthetic action. Of particular interest are the targets that have mammalian neuronal or mitochondrial orthologues such as synaptic vesicle proteins and proteins resident in lipid rafts, specialized domains of cholesterol and glyco-lipid within the larger membrane.52–54 That a protein target of anesthetic agents resides in lipid rafts provides an firm foundation upon which to speculate on the co-dependency of lipids and proteins in mediating anesthesia. Given the advances of recent years, the genetic screening approach will likely continue to yield such useful information. A more focused approach to testing the role of putative protein targets in anesthesia is by generating animals (usually mice) with mutation for genes of interest. The first transgenic technique was the generation of null mutant (so-called ‘knock-out’) animals, where expression of the target gene is completely lacking (reviewed in 55). Knock-out transgenic mice have provided compelling evidence for the role of the b3 subunit of the GABAA receptor in mediating the hypnotic effects of benzodiazepines as well as the immobilizing effects of certain inhaled anesthetic agents, an observation that illustrates quite well the discrete nature of anesthetic endpoints.55 Because knocking out the expression of an ion-channel-subunit gene may induce compensatory changes in the composition of channel subunits, network circuitry, or both, these experiments have yielded mixed results and other molecular approaches to genetic analysis have been introduced. The introduction of specific mutations into native genes (‘knock-in’) avoids the pitfalls of compensatory subunit substitution and enables an assessment of the physiologic and pharmacologic roles of specific proteins and even small regions within proteins. Ideally, such mutated receptors retain normal physiologic function yet fail to bind or respond to anesthetic agent. This methodology has already yielded exciting

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and convincing evidence for the involvement of various GABAA receptor subunits (b3 and b2) and more recently, for members of the background leak potassium channel family (TREK) in producing immobility, sedation and hypnosis.7,56 These experiments have also highlighted that the b subunits of the GABAA receptor are only partially responsible for volatile anesthetic actions, but are crucial for the actions of intravenous agents propofol and etomidate. Another important concept revealed by these knock-in experiments is that different subunits of a given receptor complex may confer discrete anesthetic effects, for example sedation versus immobility versus hypnosis (see 7 for review). This is likely explained by the presence of specific receptor subtypes in segregated neuronal circuits.57,58 Anatomic targets of volatile anesthetics The molecular actions of anesthetics cannot, in themselves, explain the mechanism of anesthesia unless they are understood within a neuroanatomic context. The observations from genetic knock-in experiments suggest that distinct combinations of receptor subunits dispersed across discrete anatomical sites underlay hypnosis, immobility and amnesia. There are therefore two facets to the anatomic localization of anesthetic actions. First because discrete functions such as movement, memory and sleep are all likely generated by similarly discrete areas of the nervous system, anesthetic modulation of those functions is also likely discretely segregated. In support of this, anesthetic agents applied within a specific anatomic region can produce behavioral endpoints that are indistinguishable from effects of systemically delivered anesthetics.59 A specific such example is anesthetic-induced immobility. Several lines of evidence make clear that volatile anesthetics ablate movement in response to painful stimuli at sites in the spinal cord.60 Although nociceptive responses can be attenuated by delivery of volatile anesthetics to the brain alone, concentrations of nearly three times MAC are required to do so.61 In contrast, amnesia is mediated by supra-spinal centers. A second facet of anatomic localization of anesthetic actions is that different anesthetic agents can act to produce identical endpoints by distinct mechanisms within the same anatomic region. For example, halothane and propofol act preferentially on dorsal horn neurons, while isoflurane acts on ventral horn neurons to produce immobility.62,63 Additionally, the molecular targets in this regions appear to be distinct for specific agents. Propofol can produce immobility via actions exclusively at GABAA receptors, while sevoflurane does so by combined effects at both GABAA and glycine receptors.64 In the brain, anatomic compartmentalization is also likely important to anesthetic actions. For example, mildly hypnotic concentrations of isoflurane reduce task-induced brain activation in some cortical regions, whereas activity in the visual cortex, motor cortex, and sub-cortical regions remains unchanged.65 Computed tomographic assessment of regional uptake of glucose in deeply anesthetized volunteers also indicates that the thalamus and midbrain reticular formation are more depressed than other regions.66 Evoked potentials traveling from the periphery to the sensory cortex show increased latency and decreased amplitude in patients under deep anesthesia with a volatile anesthetic. This signal degradation is discontinuous, occurring at specific relay sites in the thalamus and the deep cortex.67 Despite this apparent functional and anatomic segregation of anesthetic actions, some inter-play among different CNS regions likely contributes to specific behavioral

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effects. General anesthetics decrease the transmission of noxious information ascending from the spinal cord to the brain, thereby decreasing supraspinal arousal.67,68 In goats, selective delivery of general anesthetics to the torso slows cortical electroencephalographic (EEG) signals.69 Thus, ascending signals from the spinal cord affect the hypnotic actions of anesthetics in the brain, and conversely, descending signals modulate the immobilizing actions of anesthetics in the spinal cord. Lastly, an example of the degree to which anesthetics can act at discrete anatomical locales is evidenced by the ability of certain inhaled agents to mimic the protective effects of brief periods of ischemia in the heart prior to a more massive ischemic insult there.70 This ‘ischemic pre-conditioning’ property of some clinically used volatile agents is produced by actions at specific potassium channels within the myocyte mitochondria.71 More striking is that anesthetics display a dose dependency and variable efficacy with sevoflurane being the most potent.72,73 A better understanding of this astounding agent specificity in pre-conditioning may aid in a more generalized understanding of agent specificity for other effects of anesthetics. Integrated models of anesthetic actions It should be clear from the above discussion that the original unitary hypothesis of Bernard and the non-specific lipid hypothesis of Meyer and Overton have been supplanted by a wealth of data on the particular and dissociable effects of anesthetics on molecular substrates such as proteins and lipids, as well as anatomic substrates such as the brain and spinal cord. Despite the rapid advance of our molecular and neuroanatomic knowledge of these effects, the question nonetheless persists: how do volatile gases with diverse structures all give rise to the state of anesthesia? In order to address this question, we will likely need to expand our concept of anesthetic targets to include not simply structural entities (such as lipids, proteins and brain regions) but also higher-order functional processes that emerge from the temporal integration of events at these spatial structures. Behavioral integration A number of studies have attempted to relate the effects of anesthetic agents on both simple and complex neural networks to effects on higher-order systems, including behavior (see 8 for review). For example, using ex vivo preparations of hippocampus, it was found that specific subsets of neurons are differentially depressed in an agent specific manner and that the selective action of halothane on GABAA receptors was found to underlie the changes in total hippocampus synaptic activity.28,74 At a broader systemic level, inhaled anesthetic inhibition of thalamo-cortical circuits across many regions resulting in whole brain EEG slowing correlates strongly with hypnosis and amnesia in humans.75 Similar alterations between hippocampal and cortical EEG patterns have been observed in response to intravenous anesthetics.76 These observations suggest that loss of synaptic coupling between multiple cortical structures contributes to anesthesia. The question of consciousness Such higher-order levels of behavioral integration lead us to the ultimate substrate of anesthetic activity: consciousness itself. A recently hightened interest in the basis of

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consciousness has been paralleled with a renewed interest in the various anesthetic agents that modulate consciousness. The question of consciousness is intertwined with the fundamental question of general anesthesia: without understanding the mechanism of conscious representation, it is in principle impossible to understand the reversible interruption of such representation by volatile anesthetics.12 There are a variety of theories of consciousness that currently range from the philosophical (such as phenomenology) to the physical (such as quantum mechanics). While many of these theories are intellectually fascinating, it is crucial that we identify neuroscientific features of consciousness that can serve to further our understanding of anesthetic actions in an experimentally rigorous and scientifically practical manner. Although the neural correlates of consciousness are far from being elucidated, there are aspects of neural processing that appear to be necessary (although by no means sufficient) for conscious representation. The brain’s synthesis of sensory information into a unified perception is thought to be one crucial aspect of consciousness and is generally referred to as the process of ‘cognitive binding’. Cognitive binding. Cognitive binding is thought to occur at virtually all levels of cognitive processing and is thought to be a crucial event for consciousness itself.77 There have been various solutions proposed for the mechanism of cognitive binding, which can be summarized as binding by convergence, binding by assembly, and binding by synchrony. Binding by convergence denotes the process of lower-order neurons in more primary sensory areas converging upon secondary or higher-order areas for integration.78 Cells or clusters of cells have been identified that respond preferentially to particular objects, suggesting that they are attuned to synthesizing the perceptual data of an object into one representation.79,80 Binding by assembly refers to an ensemble of cells in a neural circuit whose interconnections from distinct areas of the cortex allow the integration of different features of an object. Such ensembles are thought to form in a Hebbian mechanism, i.e. their synaptic connection becomes stronger the more frequently they fire together to represent a specific perception.78 There is clear evidence for this form of cognitive binding in the brain.81,82 Finally, binding by synchrony is thought to be an important mechanism for cortical synthesis. In this schema of cognitive binding, the correlation of neurons in time is thought to provide a flexible mechanism for integrating perceptual information processed in discrete areas of the brain.83 There is abundant evidence for temporal synchronization at different scales in the brain, associated with perceptual events and tasks. Binding by synchrony is due, in part, to cortical resonance of 40 Hz with pacemaker neurons in the thalamus, and has been detected using magnetoencephalography and EEG.84,85 It should be clear that the interruption of any one of these three binding processes (convergence, assembly, and synchrony) might potentially lead to the loss of conscious representation. A number of theoretically distinct frameworks of the effects of general anesthetics on consciousness have either implicitly or explicitly suggested the inhibition of cognitive binding as an important final common mediator (see 86 for review). The effect of general anesthetics on cognitive binding processes. Various lines of evidence suggest that the mechanisms proposed to account for cognitive binding in the brain

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are affected by general anesthetics. In humans, inhaled anesthetics were seen to interrupt the EEG signature 40-Hz coherence among rostral and caudal, as well as hemispheric brain regions. This electrical decoherence was correlated with the induction and depth of anesthesia.87 In other words, the g-band synchronization that is typically observed between cortices in the conscious state was interrupted in the anesthetized state. It is important to note that anesthetics affect the coherence rather than the mere presence of g-band activity in the brain. Indeed, it has been demonstrated that general anesthetics may actually enhance 40-Hz activity in the visual system.88 This phenomenon is of clear relevance to the effects of anesthetics on conscious processing, as various forms of cognitive binding by 40-Hz oscillation have been correlated with perceptual tasks in the waking state89, aberrant hallucinatory states90, and dream states.91 Importantly, states in which 40-Hz synchrony is interrupted have been correlated with non-REM delta sleep in addition to general anesthesia.91 The interrupted 40-Hz coherence in non-REM sleep is of interest, considering the elucidation of anesthetics affecting sleep systems in the brain.92,93 Indeed, the convergence of the pathways of sleep and those mediating anesthetic effects—long of interest as a concept, but now being again pursued with renewed vigor—promises to be a productive avenue for investigating the common mechanisms of multiple states of consciousness. Binding by 40-Hz synchrony is not the only proposed mechanism of cortical integration affected by anesthetics. Studies in animals suggest that general anesthetics may inhibit oscillatory (Hebbian) assemblies in the hippocampus and the medial temporal lobe.94,95 In addition to the broader relevance of anesthetic effects on consciousness and binding, these data are directly relevant to the amnestic effects of anesthetics. Indeed, isoflurane has been shown to inhibit long-term potentiation in the hippocampus, a site of cortical integration for memory consolidation.96 In addition to anesthetic effects on binding by synchrony and Hebbian assembly, they may also effect binding by convergence of information to highly specialized neuronal cell populations. For example, isoflurane was shown to shift visual pattern recognition to component recognition a specific cortical area of nonhuman primate brain.97 It was observed that neurons in this area were still responsive to component signals in the anesthetized state but were not able to synthesize them into a complete representation. Thus, unconscious processing of elemental sensory data may persist in the anesthetized state, while the synthesis required for conscious representation is inhibited. The foregoing examples have led to the proposition of general anesthesia as a ‘cognitive unbinding’.86 More importantly, such examples reflect that processes essential for consciousness are affected by general anesthetics, and that such functional processes related to consciousness may be realistically explored as targets of anesthetics through experimentally sound methodologies. Furthermore, these functional processes are directly related to the lower-order processes at the molecular and cellular level. For example, anesthetic-induced temporal decoherence across the brain is consistent with experimental results in simpler neural networks that show synaptic desynchrony as a result of general anesthetics.76 Another example is the possibility that molecular inhibition of NMDA receptors has a direct effect in interrupting complex perceptual synthesis in the brain.98 There will likely be a rich dialogue between investigators taking a ‘top-down’ approach and those taking a ‘bottom-up’ approach.

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SUMMARY Our understanding of anesthetic mechanisms has evolved from non-specific unitary hypotheses to our current knowledge of the complex and dissociable effects of anesthetics on myriad molecular targets. Technological developments in molecular biology will no doubt enhance our ability to probe more precisely the molecular pharmacology of anesthesia. Although the current paradigm of such investigation focuses heavily on the role of protein ion channels, there are a variety of non-ion channels that are also potential targets of volatile anesthetic activity. The crucial role of lipids in neural structure and function demands that we maintain them as viable subjects of further research into general anesthetic mechanism. Persistent changes in neural gene and protein expression after general anesthesia suggest the possibility of an effect of anesthetics on neural plasticity. As stated above, the understanding of anesthetic effects on these biomolecular targets must be situated within a neuroanatomic context and the advances in neuroimaging will enhance our ability to do so. There has also been a rebirth of interest in general anesthetics for their protective effects on other organs such as the heart and kidney. Finally, anesthetic actions on molecular and anatomic structures must be appreciated in the broader perspective of the higher-order cognitive functions that are exquisitely organized in a temporal flow. As such, the fundamental mystery of general anesthesia will always be inextricably linked to the fundamental mystery of consciousness. Research agenda † identifying additional molecular targets such as ion channels or intra-cellular signaling proteins that mediate therapeutic or toxic actions of volatile anesthetics † developing experimental systems that enable a rigorous study of lipids and channel function so that the role of the lipid environment in volatile anesthetic action may be elucidated † defining the roles of specific sites in the brain and spinal cord in anesthetic actions, as well as their interactions in neural networks † identifying the neurophysiologic correlates of consciousness that may serve as functional substrates of volatile anesthetics † integrating the observations at the molecular, anatomic, and functional levels into a cohesive paradigm with explanatory power

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