Clinical Anesthesia

Chapter 5

Mechanisms of Anesthesia and Consciousness

Alex S. Evers

C. Michael Crowder

Key Points

1. The components of the anesthetic state include unconsciousness, amnesia, analgesia, immobility, and attenuation of autonomic responses to noxious stimulation.

2. Minimum alveolar concentration (MAC) remains the most robust measurement and the standard for determining the potency of volatile anesthetics.

3. Anesthetic actions on the spinal cord cannot produce either amnesia or unconsciousness. However, several lines of evidence indicate that the spinal cord is probably the site at which anesthetics act to inhibit purposeful responses to noxious stimulation.

4. A developing body of evidence indicates that inhalational anesthetics can depress the excitability of thalamic neurons, thus blocking thalamocortical communication and potentially resulting in loss of consciousness.

5. Whereas certain anesthetic effects may be attributable to specific anatomic locations (e.g., purposeful response to noxious stimulation maps to the spinal cord), existing evidence provides no basis for a single anatomic site responsible for anesthesia.

6. While current data still support the prevailing view that neuronal excitability is only slightly affected by general anesthetics, this small effect may nevertheless contribute significantly to the clinical actions of volatile anesthetics.

7. The synapse is generally thought to be the most likely relevant site of anesthetic action. Existing evidence indicates that even at this one site, anesthetics produce various effects, including presynaptic inhibition of neurotransmitter release, inhibition of excitatory neurotransmitter effect, and enhancement of inhibitory neurotransmitter effect. Furthermore, the effects of anesthetics on synaptic function differ among various anesthetic agents, neurotransmitters, and neuronal preparations.

8. Existing evidence suggests that most voltage-dependent calcium channels (VDCCs) are modestly sensitive or insensitive to anesthetics. However, some sodium channels subtypes are inhibited by volatile anesthetics and this effect may be responsible in part for a reduction in neurotransmitter release at some synapses.

9. A large body of evidence shows that clinical concentrations of many anesthetics potentiate GABA-activated currents in the central nervous system. Other members of the ligand-activated ion channel family, including glycine receptors, neuronal nicotinic receptors, and 5-HT₃receptors, are also affected by clinical concentrations of anesthetics and remain plausible anesthetic targets.

10. Activation of background K⁺ channels in mammalian vertebrates could be an important and general mechanism through which inhalational and gaseous anesthetics regulate neuronal resting membrane potential and thereby excitability.

11. Direct interactions of anesthetic molecules with proteins would not only satisfy the Meyer-Overton rule, but would also provide the simplest explanation for compounds that deviate from this rule.

12. Current evidence strongly indicates protein rather than lipid as the molecular target for anesthetic action.

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13. All anesthetic actions cannot be localized to a specific anatomic site in the central nervous system; indeed, some evidence suggests that different components of the anesthetic state may be mediated by actions at disparate anatomic sites.

14. At a molecular level, volatile anesthetics show some selectivity, but still affect the function of multiple ion channels and synaptic proteins. The intravenous anesthetics, etomidate, propofol, and barbiturates, are more specific with the GABA_A receptor as their major target.

The introduction of general anesthetics into clinical practice over 150 years ago stands as one of the seminal innovations of medicine. This single discovery facilitated the development of modern surgery and spawned the specialty of anesthesiology. Despite the importance of general anesthetics and despite more than 100 years of active research, the molecular mechanisms responsible for anesthetic action remain one of the unsolved mysteries of pharmacology.

Why have mechanisms of anesthesia been so difficult to elucidate? Anesthetics, as a class of drugs, are challenging to study for three major reasons:

1. Anesthesia, by definition, is a change in the responses of an intact animal to external stimuli. Making a definitive link between anesthetic effects observed in vitro and the anesthetic state observed and defined in vivo has proven difficult.

2. No structure–activity relationships are apparent among anesthetics; a wide variety of structurally unrelated compounds, ranging from steroids to elemental xenon, are capable of producing clinical anesthesia. This suggests that there are multiple molecular mechanisms that can produce clinical anesthesia.

3. Anesthetics work at very high concentrations in comparison to drugs, neurotransmitters, and hormones that act at specific receptors. This implies that if anesthetics do act by binding to specific receptor sites, they must bind with very low affinity and probably stay bound to the receptor for very short periods of time. Low-affinity binding is much more difficult to observe and characterize than high-affinity binding.

Despite these difficulties, molecular and genetic tools are now available that should allow for major insights into anesthetic mechanisms in the next decade. The aim of this chapter is to provide a conceptual framework for the reader to catalog current knowledge and integrate future developments about mechanisms of anesthesia. Five specific questions will be addressed in this chapter:

1. What is anesthesia and how do we measure it?

2. What is the anatomic site of anesthetic action in the central nervous system?

3. What are the cellular neurophysiologic mechanisms of anesthesia (e.g., effects on synaptic function vs. effects on action potential generation) and what anesthetic effects on ion channels and other neuronal proteins underlie these mechanisms?

4. What are the molecular targets of anesthetics?

5. How are the molecular and cellular effects of anesthetics linked to the behavioral effects of anesthetics observed in vivo?

What is Anesthesia?

General anesthesia can broadly be defined as a drug-induced reversible depression of the central nervous system (CNS) resulting in the loss of response to and perception of all external stimuli. Unfortunately, such a broad definition is inadequate for two reasons. First, the definition is not actually broad enough. Anesthesia is not simply a deafferented state; amnesia and unconsciousness are important aspects of the anesthetic state. Second, the definition is too broad, as all general anesthetics do not produce equal depression of all sensory modalities. For example, barbiturates are considered to be anesthetics, but they are not particularly effective analgesics. These conflicting problems with definition can be bypassed by a more practical description of the anesthetic state as a collection of “component” changes in behavior or perception. The components of the anesthetic state include unconsciousness, amnesia, analgesia, immobility, and attenuation of autonomic responses to noxious stimulation.

Regardless of which definition of anesthesia is used, essential to anesthesia are rapid and reversible drug-induced changes in behavior or perception. As such, anesthesia can only be defined and measured in the intact organism. Changes in behavior such as unconsciousness or amnesia can be intuitively understood in higher organisms such as mammals, but become increasingly difficult to define as one descends the phylogenetic tree. Thus, while anesthetics have effects on organisms ranging from worms to man, it is difficult to map with certainty the effects of anesthetics observed in lower organisms to any of our behavioral definitions of anesthesia. This contributes to the difficulty of using simple organisms as models in which to study the molecular mechanisms of anesthesia. Similarly, any cellular or molecular effects of anesthetics observed in higher organisms can be extremely difficult to link with the constellation of behaviors that constitute the anesthetic state. The absence of a simple and concise definition of anesthesia is clearly one of the stumbling blocks to elucidating the mechanisms of anesthesia at a molecular and cellular level.

An additional difficulty in defining anesthesia is that our understanding of the mechanisms of consciousness is rather amorphous at present. One cannot easily define anesthesia when the neurobiological phenomena ablated by anesthesia are not well understood. Nevertheless, recent advances in the study of sleep and attention have identified what may form the anatomic and neurophysiological basis for sleep and perhaps other forms of unconsciousness.¹ Central to the mechanism of sleep is a set of hypothalamic nuclei that appear to form an awake/sleep switch mechanism (Fig. 5-1). The ventrolateral preoptic nucleus (VLPO) in the anterior hypothalamus promotes sleep while the tuberomammillary nucleus (TMN) in the posterior hypothalamus promotes wakefulness. Importantly, the VLPO and the TMN are mutually inhibitory. Thus. for example, if by influence of other modulatory sleep-promoting nuclei the activity of the VLPO gains ground relative to the TMN, the VLPO will ultimately shut down the output of the TMN and sleep will be favored. On the other hand during wakefulness, the TMN is dominant and silences the VLPO. Modulatory influences on the TMN and VLPO include orexinergic neurons in the lateral hypothalamus, the circadian clock, which is directly modulated by light and contained within the hypothalamic suprachiasmatic nucleus, and multiple brainstem nuclei, in particular the locus coeruleus and dorsal raphe. These brainstem nuclei as a whole promote arousal and are a part of the reticular activating formation. In addition to synaptic modulators, adenosine has been proposed as a neurohumoral factor that promotes sleep by disinhibiting the VLPO. The TMN and the VLPO are thought to promote the awake or sleep state by acting on thalamic and cortical circuits, either directly or through the reticular activating formation. The thalamus and cortex maintain wakefulness and consciousness through complex interactions that may involve

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intrinsic oscillators and widespread synaptic communication. Awareness and consciousness is thought to emerge from communication between the prefrontal cortex and multiple cortical and subcortical areas that have distributed representations of a perception. Again, the precise mechanisms of the emergent properties of consciousness are unclear. As discussed later, some recent evidence implicates components of the sleep switch as anatomic targets of certain general anesthetics.

Figure 5-1. Simplified sleep/wake control circuit. The wake/sleep switch is composed of the mutually-inhibitory ventrolateral preoptic nucleus (VLPO) and the tuberomammillary nucleus (TMN) hypothalamic neurons. The direction of this switch is influenced by humoral factors such as adenosine, the circadian clock, other hypothalamic neurons releasing orexin (not shown), and brainstem arousal nuclei such as the dorsal raphe (DR) and the locus coeruleus (LC). Both the wake/sleep switch and the brainstem arousal system act on higher order circuits in the thalamus and cerebral cortex. General anesthetics appear to act on multiple components of the sleep/wake control system. 5-HT, 5-hydroxytryptamine/serotonin; Gal, galanin; NE, norepinephrine; His, histamine; GABA, γ-aminobutyric acid.

How is Anesthesia Measured?

In order to study the pharmacology of anesthetic action, quantitative measurements of anesthetic potency are absolutely essential. To this end, Quasha and colleagues² have defined the concept of MAC, or minimum alveolar concentration. MAC is defined as the alveolar partial pressure of a gas at which 50% of humans do not respond to a surgical incision. In animals, MAC is defined as the alveolar partial pressure of a gas at which 50% of animals do not respond to a noxious stimulus, such as tail clamp,³ or at which they lose their righting reflex. The use of MAC as a measure of anesthetic potency has two major advantages. First, it is an extremely reproducible measurement that is remarkably constant over a wide range of species.² Second, the use of end-tidal gas concentration provides an index of the “free” concentration of drug required to produce anesthesia since the end-tidal gas concentration is in equilibrium with the free concentration in plasma. The MAC concept has several important limitations, particularly when trying to relate MAC values to anesthetic potency observed in vitro. First, the end point in a MAC determination is quantal: a subject is either anesthetized or unanesthetized; it cannot be partially anesthetized. Furthermore, MAC represents the average response of a whole population of subjects rather than the response of a single subject. The quantal nature of the MAC measurement makes it very difficult to compare MAC measurements to concentration–response curves obtained in vitro, where the graded response of a single preparation is measured as a function of anesthetic concentration. The second limitation of MAC measurements is that they can only be directly applied to anesthetic gases. Parenteral anesthetics (barbiturates, neurosteroids, propofol) cannot be assigned a MAC value, making it difficult to compare the potency of parenteral and volatile anesthetics. A MAC equivalent for parental anesthetics is the free concentration of the drug in plasma required to prevent response to a noxious stimulus in 50% of subjects; this value has been estimated for several parenteral anesthetics.⁴ A third limitation of MAC is that it is highly dependent on the anesthetic end point used to define it. For example, if loss of response to a verbal command is used as an anesthetic end point, the MAC values obtained (MAC_awake) will be much lower than classic MAC values based on response to a noxious stimulus. Indeed, each behavioral component of the anesthetic state will likely have a different MAC value. Despite its limitations, MAC remains the most robust measurement and the standard for determining the potency of volatile anesthetics.

Because of the limitations of MAC, monitors that measure some correlate of anesthetic depth have been introduced into clinical practice.⁵ The most popular of these monitors converts spontaneous electroencephalogram waveforms into a single value that correlates with anesthetic depth for some general anesthetics. Anesthetic depth monitors have great potential. They may reduce the incidence of awareness during anesthesia, which is estimated to be approximately 0.1 to 0.2%.⁶ They may also reduce the amount of anesthetic used and may hasten emergence and recovery room discharge. However, at this time whether any of the available anesthetic depth monitors is superior to MAC, to standardized dosing of intravenous anesthetics, or to clinical indicators of anesthetic depth is controversial and is still an active area of investigation.

Where in the Central Nervous System do Anesthetics Work?

In principle, general anesthesia could result from interruption of nervous system activity at myriad levels. Plausible targets include peripheral sensory receptors, spinal cord, brainstem, and cerebral cortex. Of these potential sites, only peripheral sensory receptors can be eliminated as an important site of anesthetic action. Animal studies have shown that fluorinated volatile anesthetics have no effect on cutaneous mechanosensors in cats⁷ and can even sensitize nociceptors in monkeys.⁸ Furthermore, selective perfusion studies in dogs have shown that MAC for isoflurane is unaffected by the presence or absence of isoflurane at the site of noxious stimulation, provided that the CNS is perfused with blood containing isoflurane.⁹

Spinal Cord

Clearly, anesthetic actions on the spinal cord cannot produce either amnesia or unconsciousness. However, several lines of evidence indicate that the spinal cord is probably the site at

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which anesthetics act to inhibit purposeful responses to noxious stimulation. This is, of course, the end point used in most measurements of anesthetic potency. Rampil and colleagues^10,11 have shown that MAC values for fluorinated volatile anesthetics are unaffected in the rat by either decerebration¹⁰ or cervical spinal cord transection.¹¹ Antognini and Schwartz¹² have used the strategy of isolating the cerebral circulation of goats to explore the contribution of brain and spinal cord to the determination of MAC. They found that when isoflurane is administered only to the brain, MAC is 2.9%, whereas when it is administered to the entire body, MAC is 1.2%. Surprisingly, when isoflurane was preferentially administered to the body and not to the brain, isoflurane MAC was reduced to 0.8%.¹³ The actions of volatile anesthetics in the spinal cord are mediated, at least in part, by direct effects on the excitability of spinal motor neurons. This conclusion has been substantiated by experiments in rats,¹⁴ goats,¹⁵ and humans¹⁶ showing that volatile anesthetics depress the amplitude of the F wave in evoked potential measurements (F-wave amplitude correlates with motor neuron excitability). These provocative results suggest not only that anesthetic action at the spinal cord underlies MAC, but also that anesthetic action on the brain may actually sensitize the cord to noxious stimuli. The plausibility of the spinal cord as a locus for anesthetic immobilization is also supported by several electrophysiological studies showing inhibition of excitatory synaptic transmission in the spinal cord.^17,18,19,20

Brainstem, Hypothalamic, and Thalamic Arousal Systems

The reticular activating system, a diffuse collection of brainstem neurons involved in arousal behavior, has long been speculated to be a site of general anesthetic action on consciousness. Evidence to support this notion came from early whole-animal experiments showing that electrical stimulation of the reticular activating system could induce arousal behavior in anesthetized animals.²¹ A role for the brainstem in anesthetic action is also supported by studies examining somatosensory evoked potentials. Generally, these studies show that anesthetics produce increased latency and decreased amplitude of cortical potentials, indicating that anesthetics inhibit information transfer through the brainstem.²² In contrast, studies using brainstem auditory evoked potentials have shown variable effects ranging from depression to enhancement of information transfer through the reticular formation.^23,24,25 While there is evidence that the reticular formation of the brainstem is a locus for anesthetic effects, it cannot be the only anatomic site of anesthetic action for two reasons. First, as discussed, the brainstem is not even required for anesthetics to inhibit responsiveness to noxious stimuli. Second, the reticular formation can be largely ablated without eliminating awareness.²⁶

Within the reticular formation is a set of pontine noradrenergic neurons called the locus coeruleus. The locus coeruleus widely innervates targets in the cortex, thalamus, and hypothalamus including the sleep-promoting VLPO. As discussed previously, the mutually inhibitory VLPO and TMN may form a sleep/awake switch circuit. This switch was directly implicated in anesthetic action by a set of elegant experiments from Nelson et al.²⁷ They showed that the application of a GABAergic antagonist directly onto the TMN diminished the efficacy of the anesthetics propofol and pentobarbital. Indeed, discrete application of the GABAergic antagonist gabazine onto the TMN markedly reduced the duration of sedation produced by systemically administered propofol or pentobarbital. This effect is unlikely to be a consequence of a nonspecific increase in arousal state because systemically administered gabazine did not antagonize the potency of ketamine whereas it did antagonize propofol and pentobarbital in a manner similar to application directly onto the TMN. This result strongly implicates the VLPO/TMN sleep switch as a site for the sedative action of GABAergic anesthetics like propofol and barbiturates. However, general anesthesia is clearly not equivalent to sleep. By definition, one cannot be aroused from general anesthesia. Thus, additional neuroanatomical loci besides those mediating sleep are likely to be targeted. One area of the brain that has been postulated as a potential site of anesthetic action is the thalamus. The thalamus is important in relaying sensory modalities and motor information to the cortex via thalamocortical pathways. A developing body of evidence indicates that inhalational anesthetics can depress the excitability of thalamic neurons, thus blocking thalamocortical communication and potentially resulting in loss of consciousness.

Cerebral Cortex

The cerebral cortex is the major site for integration, storage, and retrieval of information. As such, it is a likely site at which anesthetics might interfere with complex functions like memory and awareness. Anesthetics clearly alter cortical electrical activity, as evidenced by the changes in surface electroencephalogram patterns recorded during anesthesia. Anesthetic effects on patterns of cortical electrical activity vary widely among anesthetics,²⁸ providing an initial suggestion that all anesthetics are not likely to act through identical mechanisms. More detailed in vitro electrophysiological studies examining anesthetic effects on different cortical regions support the notion that anesthetics can differentially alter neuronal function in various cortical preparations. For example, volatile anesthetics have been shown to inhibit excitatory transmission at some synapses in the olfactory cortex²⁹ but not at others.³⁰ Similarly, whereas volatile anesthetics inhibit excitatory transmission in the dentate gyrus of the hippocampus,³¹ these same drugs can actually enhance excitatory transmission at other synapses in the hippocampus.³² Anesthetics also produce a variety of effects on inhibitory transmission in the cortex. A variety of parenteral and inhalation anesthetics have been shown to enhance inhibitory transmission in olfactory cortex³⁰ and in the hippocampus.³³ Conversely, volatile anesthetics have also been reported to depress inhibitory transmission in hippocampus.³⁴

Summary

Anesthetics produce effects on a variety of anatomic structures in the CNS, including spinal cord, brainstem, hypothalamus, and cerebral cortex. Whereas certain anesthetic effects may be attributable to specific anatomic locations (e.g., purposeful response to noxious stimulation maps to the spinal cord), existing evidence provides no basis for a single anatomic site responsible for anesthesia. This difficulty in identifying a site for anesthesia might plausibly result from the various components of the anesthetic state being produced by anesthetic effects on different regions of the CNS. Nevertheless, despite the difficulty in identifying a common anatomic site for anesthesia, investigators have continued to look for other unifying principles in anesthetic action. Specifically, attention has been focused on identifying common cellular or molecular anesthetic targets that may have a wide anatomic distribution, explaining the ability of anesthetic to affect nervous system function in an anatomically diffuse manner.

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How do Anesthetics Interfere with the Electrophysiologic Function of the Nervous System?

In the simplest terms anesthetics inhibit or “turn off” vital CNS functions. They must do this by acting at specific physiologic “switches.” A great deal of investigative effort has been devoted to identifying these switches. In principle, the CNS could be switched off by several means:

1. By depressing those neurons or pattern generators that subserve a pacemaker function in the CNS.

2. By reducing overall neuronal excitability, either by changing resting membrane potential or by interfering with the processes involved in generating an action potential.

3. By reducing communication between neurons; specifically, by either inhibiting excitatory synaptic transmission or enhancing inhibitory synaptic transmission.

Pattern Generators

Information concerning the effects of anesthetics on pattern-generating neuronal circuits in the CNS is limited, but clinical concentrations of anesthetics are likely to have significant effects on these circuits. The simplest evidence for this is the observation that most anesthetics exert profound effects on respiratory rate and rhythm, strongly suggesting an effect on respiratory pattern generators in the brainstem. Invertebrate studies suggest that volatile anesthetics can selectively inhibit the spontaneous (pacemaker) firing of specific neurons. As shown in Figure 5-2, halothane (1 MAC) completely inhibits spontaneous action potential generation by one neuron in the right parietal ganglion of the great pond snail while producing no observable effect on the firing frequency of adjacent neurons.³⁵

Neuronal Excitability

The ability of a neuron to generate an action potential is determined by three parameters: resting membrane potential, the threshold potential for action potential generation, and the function of voltage-gated sodium channels. Anesthetics can hyperpolarize (create a more negative resting membrane potential) both spinal motor neurons and cortical neurons,^36,37 and this ability to hyperpolarize neurons correlates with anesthetic potency. In general, the increase in resting membrane potential produced by anesthetics is small in magnitude and is unlikely to have an effect on axonal propagation of an action potential. Small changes in resting potential may, however, inhibit the initiation of an action potential either at a postsynaptic site or in a spontaneously firing neuron. Indeed, hyperpolarization is responsible for the inhibition of spontaneous action potential generation shown in Figure 5-2. Recent evidence also indicates that isoflurane hyperpolarizes thalamic neurons, leading to an inhibition of tonic firing of action potentials.³⁸ There is no evidence indicating that anesthetics alter the threshold potential of a neuron for action potential generation. However, the data are conflicting on whether the size of the action potential, once initiated, is diminished by general anesthetics. A classic article by Larabee and Posternak³⁹ demonstrated that concentrations of ether and chloroform that completely block synaptic transmission in mammalian sympathetic ganglia have no effect on presynaptic action potential amplitude. Similar results have been obtained with fluorinated volatile anesthetics in mammalian brain preparations.^29,31 This dogma that the action potential is relatively resistant to general anesthetics has been challenged by more recent reports that volatile anesthetics at clinical concentrations produce a small but significant reduction in the size of the action potential in mammalian neurons.^40,41 In one case, the reduction in the action potential was shown to be amplified at the presynaptic terminal resulting in a large reduction in neurotransmitter release.⁴¹ Thus, while current data still support the prevailing view that neuronal excitability is only slightly affected by general anesthetics, this small effect may nevertheless contribute significantly to the clinical actions of volatile anesthetics.

Figure 5-2. Selectivity of volatile anesthetic inhibition of neuronal automaticity. A: Halothane (1 MAC) reversibly inhibits the spontaneous firing activity of a neuron from the parietal ganglion of Lymnaea stagnalis). B: The same concentration of halothane has no effect on the firing activity of an adjacent, and apparently identical, neuron). Note that in A, halothane markedly reduces resting membrane potential in addition to inhibiting firing. (Reprinted with permission from Franks NP, Lieb WR: Mechanisms of general anesthesia. Environ Health Perspect 87:204, 1990.)

Synaptic Function

Synaptic function is widely considered to be the most likely subcellular site of general anesthetic action. Neurotransmission across both excitatory and inhibitory synapses is markedly altered by general anesthetics. General anesthetics inhibit excitatory synaptic transmission in a variety of preparations, including sympathetic ganglia,³⁹ olfactory cortex,²⁹

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hippocampus,³¹ and spinal cord.¹⁹ However, not all excitatory synapses appear to be equally sensitive to anesthetics; indeed, transmission across some hippocampal excitatory synapses is enhanced by inhalational anesthetics.³² In a similar fashion, general anesthetics both enhance and depress inhibitory synaptic transmission in various preparations. In a classic article in 1975, Nicoll et al⁴² showed that barbiturates enhanced inhibitory synaptic transmission by prolonging the decay of the GABAergic inhibitory postsynaptic current. Enhancement of inhibitory transmission has also been observed with many other general anesthetics including etomidate,⁴³ propofol,⁴⁴ inhalational anesthetics,³⁰ and neurosteroids.⁴⁵Although anesthetic enhancement of inhibitory currents has received a great deal of attention as a potential mechanism of anesthesia,⁴ it is important to note that there is also a large body of experimentation showing that clinical concentrations of general anesthetics can depress inhibitory postsynaptic potentials in hippocampus^34,46,47 and in spinal cord.²⁰Anesthetics do appear to have preferential effects on synapses, but there is a great deal of heterogeneity in the manner in which anesthetic agents affect different synapses. This is not surprising given the large variation in synaptic structure, function (i.e., efficacy), and chemistry (neurotransmitters, modulators) extant in the nervous system.

Presynaptic Effects

General anesthetics affect synaptic transmission both pre- and postsynaptically. However, the magnitude and even the type of effect vary according to the type of synapse and the particular anesthetic. Presynaptically, neurotransmitter release from glutamatergic synapses has consistently been found to be inhibited by clinical concentrations of volatile anesthetics. For example, a study by Perouansky and colleagues⁴⁸ conducted in mouse hippocampal slices showed that halothane inhibited excitatory postsynaptic potentials elicited by presynaptic electrical stimulation, but not those elicited by direct application of glutamate. This indicates that halothane must be acting to prevent the release of glutamate, the major excitatory neurotransmitter in the brain. MacIver and colleagues extended these observations by finding that the inhibition of glutamate release from hippocampal neurons is not due to effects at GABAergic synapses that could indirectly decrease transmitter release from glutamatergic neurons.³² Effects of intravenous anesthetics on glutamate release have also been demonstrated, but the evidence is more limited and the effects potentially indirect.^49,50 The data for anesthetic effects on inhibitory neurotransmitter release is mixed. Inhibition,⁵¹ stimulation,^52,53 and no effect⁵⁴ have been reported for volatile anesthetic and intravenous anesthetic action on GABA (γ-aminobutyric acid) release. In a brain synaptosomal preparation where effects on both GABA and glutamate release could be studied simultaneously, Westphalen and Hemmings⁵⁵ found that glutamate and, to a lesser degree, GABA release were inhibited by clinical concentrations of isoflurane. The mechanism underlying anesthetic effects on transmitter release has not been established. The effects of anesthetics on neurotransmitter release do not appear to be mediated by reduced neurotransmitter synthesis or storage, but rather by a direct effect on the process of neurosecretion. A variety of evidence argues that at some synapses a substantial portion of the anesthetic effect is upstream of the transmitter release machinery, perhaps on presynaptic sodium channels or potassium leak channels (see later discussion). However, genetic data in Caenorhabditis elegans shows that the transmitter release machinery strongly influences volatile anesthetic sensitivity⁵⁶; at present, it is unclear whether these findings represent species differences or different aspects of the same mechanism.

Postsynaptic Effects

Anesthetics alter the postsynaptic response to released neurotransmitter. The effects of general anesthetics on excitatory neurotransmitter receptor function vary depending on neurotransmitter type, anesthetic agent, and preparation. Richards and Smaje⁵⁷ examined the effects of several anesthetic agents on the response of olfactory cortical neurons to application of glutamate, the major excitatory neurotransmitter in the CNS. They found that while pentobarbital, diethyl ether, methoxyflurane, and alphaxalone depressed the electrical response to glutamate, halothane was without effect. In contrast, when acetylcholine was applied to the same olfactory cortical preparation, halothane and methoxyflurane stimulated the electrical response whereas pentobarbital had no effect; only alphaxalone depressed the electrical response to acetylcholine.⁵⁸ The effects of anesthetics on neuronal responses to inhibitory neurotransmitters are more consistent. A wide variety of anesthetics, including barbiturates, etomidate, neurosteroids, propofol, and the fluorinated volatile anesthetics, have been shown to enhance the electrical response to exogenously applied GABA (for a review, see ref. 59). For example, Figure 5-3 illustrates the ability of enflurane to increase both the amplitude and the duration of the current elicited by application of GABA to hippocampal neurons.⁶⁰

Summary

Attempts to identify a physiologic switch at which anesthetics act have suffered from their own success. Anesthetics produce a variety of effects on many physiologic processes that might logically contribute to the anesthetic state, including neuronal automaticity, neuronal excitability, and synaptic function. The synapse is generally thought to be the most likely relevant site of anesthetic action. Existing evidence indicates that even at this one site, anesthetics produce various effects, including presynaptic inhibition of neurotransmitter release, inhibition of excitatory neurotransmitter effect, and enhancement of inhibitory neurotransmitter effect. Furthermore, the effects of anesthetics on synaptic function differ among various anesthetic agents, neurotransmitters, and neuronal preparations.

Anesthetic Actions on Ion Channels

Ion channels are one likely target of anesthetic action. The advent of patch clamp techniques in the early 1980s made it possible to directly measure the currents from single ion channel proteins. It was attractive to think that anesthetic effects on a small number of ion channels might help to explain the complex physiologic effects of anesthetics that we have already described. Accordingly, during the 1980s and 1990s a major effort was directed at describing the effects of anesthetics on the various kinds of ion channels. The following section summarizes and distills this effort. For the purposes of this discussion, ion channels are cataloged according to the stimuli to which they respond by opening or closing (i.e., their mechanism of gating).

Anesthetic Effects on Voltage-Dependent Ion Channels

A variety of ion channels can sense a change in membrane potential and respond by either opening or closing their pore. These channels include voltage-dependent sodium, potassium,

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and calcium channels, all of which share significant structural homologies. Voltage-dependent sodium and potassium channels are largely involved in generating and shaping action potentials. The effects of anesthetics on these channels have been extensively studied by Haydon and Urban⁶¹ in the squid giant axon. These studies show that these invertebrate sodium channels and potassium channels are remarkably insensitive to volatile anesthetics. For example, 50% inhibition of the peak sodium channel current required halothane concentrations 8 times those required to produce

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anesthesia. The delayed rectifier potassium channel was even less sensitive, requiring halothane concentrations more than 20 times those required to produce anesthesia. Similar results have been obtained in a mammalian cell line (GH₃ pituitary cells) where both sodium and potassium currents were inhibited by halothane only at concentrations greater than 5 times those required to produce anesthesia.⁶² However, a number of recent studies with volatile anesthetics have challenged the notion that voltage-dependent sodium channels are insensitive to anesthetics. Rehberg and colleagues⁶³ expressed rat brain IIA sodium channels in a mammalian cell line, and showed that clinically relevant concentrations of a variety of inhalational anesthetics suppressed voltage-elicited sodium currents. Ratnakumari and Hemmings⁶⁴ showed that sodium flux mediated by rat brain sodium channels was significantly inhibited by clinical concentrations of halothane. Shiraishi and Harris⁶⁵ documented the effects of isoflurane on a variety of sodium channel subtypes and found that several but not all subtypes are sensitive to clinical concentrations. Finally, as previously described, in a rat brainstem neuron, Wu and colleagues⁴¹ found that a small inhibition of sodium currents by isoflurane resulted in a large inhibition of synaptic activity. Thus, sodium channel activity not only appears to be inhibited by volatile anesthetics, but this inhibition results in a significant reduction in synaptic function, at least at some mammalian synapses. Intravenous anesthetics have also been shown to inhibit sodium channels, but the concentrations for this effect are supra-clinical.^66,67

Figure 5-3. Enflurane potentiates the ability of GABA (γ-aminobutyric acid) to activate a chloride current in cultured rat hippocampal cells. This potentiation is rapidly reversed by removal of enflurane (wash; A). Enflurane increases both the amplitude of the current (B) and the time (τ1/2) it takes for the current to decay (C). (Reproduced with permission from Jones MV, Brooks PA, Harrison L: Enhancement of γ-aminobutyric acid-activated Cl- currents in cultured rat hippocampal neurones by three volatile anaesthetics. J Physiol 449:289, 1992.)

Voltage-dependent calcium channels (VDCCs) serve to couple electrical activity to specific cellular functions. In the nervous system, VDCCs located at presynaptic terminals respond to action potentials by opening. This allows calcium to enter the cell, activating calcium-dependent secretion of neurotransmitter into the synaptic cleft. At least six types of calcium channels (designated L, N, P, Q, R, and T) have been identified on the basis of electrophysiological properties and a larger number based on amino acid sequence similarities. N-, P-, Q-, and R-type channels, as well as some of the untitled channels, are preferentially expressed in the nervous system and are thought to play a major role in synaptic transmission. L-type calcium channels, although expressed in brain, have been best studied in their role in excitation–contraction coupling in cardiac, skeletal, and smooth muscle and are thought to be less important in synaptic transmission. The effects of anesthetics on L- and T-type currents have been well characterized,^62,68,69 and there are some reports concerning the effects of anesthetics on N- and P-type currents.^70,71,72 As a general rule, these studies have shown that volatile anesthetics inhibit VDCCs (50% reduction in current) at concentrations 2 to 5 times those required to produce anesthesia in humans, with less than a 20% inhibition of calcium current at clinical concentrations of anesthetics. However, some studies have found VDCCs that are extremely sensitive to anesthetics. Takenoshita and Steinbach⁷³ reported a T-type calcium current in dorsal root ganglion neurons that was inhibited by subanesthetic concentrations of halothane. Additionally, ffrench-Mullen and colleagues⁷⁴ have reported a VDCC of unspecified type in guinea pig hippocampus that is inhibited by pentobarbital at concentrations identical to those required to produce anesthesia. Thus, VDCCs could well mediate some actions of general anesthetics, but their general insensitivity makes them unlikely to be major targets.

Potassium channels are the most diverse of the ion channels types and include voltage-gated, background or leak channels that open over a wide-range of voltages including at the resting membrane potential of neurons, second messenger and ligand-activated, and so-called inward rectifying channels; some channels fall into more than one category. High concentrations of both volatile anesthetics and intravenous anesthetics are required to affect significantly the function of voltage-gated K⁺ channels.^61,75,76 Similarly, classic inward rectifying K⁺ channels are relatively insensitive to sevoflurane and barbiturates.^77,78,79 However, some background K⁺ channels are quite sensitive to volatile anesthetics.

Summary

Existing evidence suggests that most VDCCs are modestly sensitive or insensitive to anesthetics. However, some sodium channels subtypes are inhibited by volatile anesthetics and this effect may be responsible in part for a reduction in neurotransmitter release at some synapses. Additional experimental data will be required to establish whether anesthetic-sensitive VDCCs are localized to specific synapses at which anesthetics have been shown to inhibit neurotransmitter release.

Anesthetic Effects on Ligand-Gated Ion Channels

Fast excitatory and inhibitory neurotransmission is mediated by the actions of ligand-gated ion channels. Synaptically released glutamate or GABA diffuse across the synaptic cleft and bind to channel proteins that open as a consequence of neurotransmitter release. The channel proteins that bind GABA (GABA_A receptors) are members of a superfamily of structurally related ligand-gated ion channel proteins that include nicotinic acetylcholine receptors, glycine receptors, and 5-HT₃ receptors. Based on the structure of the nicotinic acetylcholine receptor, each ligand-gated channel is thought to be composed of five nonidentical subunits. The glutamate receptors also comprise a family, each receptor thought to be a tetrameric protein composed of structurally related subunits. The ligand-gated ion channels provide a logical target for anesthetic action because selective effects on these channels could inhibit fast excitatory synaptic transmission and/or facilitate fast inhibitory synaptic transmission. The effects of anesthetic agents on ligand-gated ion channels are thoroughly cataloged in a review by Krasowski and Harrison.⁵⁹ The following section provides a brief summary of this large body of work.

Glutamate-Activated Ion Channels

Glutamate-activated ion channels have been classified, based on selective agonists, into three categories: AMPA receptors, kainate receptors, and NMDA receptors. AMPA and kainate receptors are relatively nonselective monovalent cation channels involved in fast excitatory synaptic transmission, whereas NMDA channels conduct not only Na⁺ and K⁺ but also Ca⁺⁺and are involved in long-term modulation of synaptic responses (long-term potentiation). Studies from the early 1980s in mouse and rat brain preparations showed that AMPA- and kainate-activated currents are insensitive to clinical concentrations of halothane,⁸⁰ enflurane,⁸¹ and the neurosteroid allopregnanolone.⁸² In contrast, kainate- and AMPA-activated currents were shown to be sensitive to barbiturates; in rat hippocampal neurons, 50 µM pentobarbital (pentobarbital produces anesthesia at approximately 50 µM) inhibited kainate and AMPA responses by 50%.⁸² More recent studies using cloned and expressed glutamate receptor subunits show that submaximal agonist responses of GluR3 (AMPA-type) receptors are inhibited by fluorinated volatile anesthetics whereas agonist responses of GluR6 (kainate-type) receptors are enhanced.⁸³ In contrast both GluR3 and GluR6 receptors are inhibited by pentobarbital. The directionally opposite effects of the volatile anesthetics on different glutamate receptor subtypes may explain the earlier inconclusive effects observed in tissue, where multiple subunit types are expressed. These opposite effects have also been used as a strategy to identify critical sites on the molecules involved in anesthetic effect. By producing GluR3/GluR6 receptor chimeras (receptors made up of various combinations of sections of the GluR3 and GluR6 receptors) and screening for volatile anesthetic effect, specific areas of the protein required for volatile anesthetic potentiation of GluR6 have been identified. Subsequent site-directed mutagenesis studies have identified a specific glycine residue (Gly-819) as critical for volatile anesthetic action on GluR6-containing receptors.⁸⁴

NMDA-activated currents also appear to be sensitive to a subset of anesthetics. Electrophysiological studies show virtually no effects of clinical concentrations of volatile anesthetics,^80,81 neurosteroids, or barbiturates⁸² on NMDA-activated currents. It should be noted that there is some evidence from flux studies that volatile anesthetics may inhibit NMDA-activated channels. A study in rat brain microvesicles showed that anesthetic concentrations (0.2 to 0.3 mM) of halothane and enflurane inhibited NMDA-activated calcium flux by 50%.⁸⁵ In contrast, ketamine is a potent and selective inhibitor of NMDA-activated currents. Ketamine stereoselectively inhibits NMDA currents by binding to the phencyclidine site on the NMDA receptor protein.^86,87,88 The anesthetic effects of ketamine in intact animals show the same stereoselectivity as that observed in vitro,⁸⁹ suggesting that the NMDA receptor may be the principal molecular target for the anesthetic actions of ketamine. Two other recent findings suggest that NMDA receptors may be an important target for nitrous oxide and xenon. These studies show that N₂O^90,91 and xenon⁹² are potent and selective inhibitors of NMDA-activated currents. This is illustrated in Figure 5-4, showing that N₂O inhibits NMDA-elicited, but not GABA-elicited, currents in hippocampal neurons.

GABA-Activated Ion Channels

GABA is the most important inhibitory neurotransmitter in the mammalian CNS. GABA-activated ion channels (GABA_A receptors) mediate the postsynaptic response to synaptically released GABA by selectively allowing chloride ions to enter and thereby hyperpolarizing neurons. GABA_A receptors are multi-subunit proteins consisting of various combinations of α, β, δ and ε subunits, and there are many subtypes of each of these subunits. The function of GABA_A receptors is modulated by a wide variety of pharmacologic agents including convulsants, anticonvulsants, sedatives, anxiolytics, and anesthetics.⁹³ The effects of these various drugs on GABA_A receptor function varies across brain regions and cell types. The following section briefly reviews the effects of anesthetics on GABA_A receptor function.

Barbiturates, anesthetic steroids, benzodiazepines, propofol, etomidate, and the volatile anesthetics all modulate GABA_A receptor function.^{60,93,94,95,96} These drugs produce three kinds of effects on the electrophysiological behavior of the GABA_A receptor channels: potentiation, direct gating, and inhibition. Potentiation refers to the ability of anesthetics to increase markedly the current elicited by low concentrations of GABA, but to produce

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no increase in the current elicited by a maximally effective concentration of GABA. Potentiation is illustrated in Figure 5-5, showing the effects of halothane on currents elicited by a range of GABA concentrations in dissociated cortical neurons. Anesthetic potentiation of GABA_A currents generally occurs at concentrations of anesthetics within the clinical range.Direct gating refers to the ability of anesthetics to activate GABA_A channels in the absence of GABA. Generally, direct gating of GABA_A currents occurs at anesthetic concentrations higher than those used clinically, but the concentration–response curves for potentiation and for direct gating can overlap. It is not known whether direct gating of GABA_A channels is either required for or contributes to the effects of anesthetics on GABA-mediated inhibitory synaptic transmission in vivo. In the case of anesthetic steroids, strong evidence indicates that potentiation, rather than direct gating of GABA_A currents, is required for producing anesthesia.⁹⁷ Anesthetics can also inhibit GABA-activated currents. Inhibition refers to the ability of anesthetics to prevent GABA from initiating current flow through GABA_A channels, and has generally been observed at high concentrations of both GABA and anesthetic.^98,99Inhibition of GABA_A channels may help to explain why volatile anesthetics have, in some cases, been observed to inhibit rather than facilitate inhibitory synaptic transmission.³⁴

Figure 5-4. Nitrous oxide inhibits NMDA-elicited, but not GABA-elicited, currents in rat hippocampal neurons. A: Eighty percent N2O has no effect on holding current (upper trace), but inhibits the current elicited by NMDA. B: N2O causes a rightward and downward shift of the NMDA concentration–response curve, indicating a mixed competitive/noncompetitive antagonism. C: Eighty percent N2O has little effect on GABA-elicited currents. In contrast, an equipotent anesthetic concentration of pentobarbital markedly enhances the GABA-elicited current. (Reproduced with permission from Jevtovic-Todorovic V, Todorovic SM, Mennerick S et al: Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant, and neurotoxin. Nat Med 4:460, 1998.)

Figure 5-5. The effects of halothane (Hal), enflurane (Enf), and fluorothyl (HFE) on GABA-activated chloride currents in dissociated rat CNS neurons. A: Clinical concentrations of halothane and enflurane potentiate the ability of GABA to elicit a chloride current. The convulsant fluorothyl antagonizes the effects of GABA (γ-aminobutyric acid. B: GABA causes a concentration-dependent activation of a chloride current. Halothane shifts the GABA concentration–response curve to the left (increases the apparent affinity of the channel for GABA), whereas fluorothyl shifts the curve to the right (decreases the apparent affinity of the channel for GABA). (Reproduced with permission from Wakamori M, Ikemoto Y, Akaike N: Effects of two volatile anesthetics and a volatile convulsant on the excitatory and inhibitory amino acid responses in dissociated CNS neurons of the rat. J Neurophysiol 66:2014, 1991.)

Effects of anesthetics have also been observed on the function of single GABA_A channels. These studies show that barbiturates,⁹⁴ propofol,⁹⁶ and volatile anesthetics¹⁰⁰ do not alter the conductance (rate at which ions traverse the open channel) of the channel, but that they increase the frequency with which the channel opens and/or the average length of time that the channel remains open. Collectively, the whole cell and single channel data are most consistent with the idea that clinical concentrations of anesthetics produce a change in the conformation of GABA_A receptors that increases the affinity of the receptor for GABA. This is consistent with the ability of anesthetics to increase the duration of inhibitory postsynaptic potentials, since higher affinity binding of GABA would slow the dissociation of GABA from postsynaptic GABA_A channels. It would not be expected that anesthetics would increase the peak amplitude of a GABAergic inhibitory postsynaptic potential since synaptically

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released GABA probably reaches very high concentrations in the synapse. Higher concentrations of anesthetics can produce additional effects, either directly activating or inhibiting GABA_A channels. Consistent with these ideas, a study by Banks and Pearce¹⁰¹ showed that isoflurane and enflurane simultaneously increased the duration and decreased the amplitude of GABAergic inhibitory postsynaptic currents in hippocampal slices.

Despite the similar effects of many anesthetics on GABA_A receptor function, there is significant evidence that the various anesthetics do not act by binding to a single common binding site on the channel protein. First, even anesthetics that directly activate the channel probably do not bind to the GABA binding site. This is most clearly demonstrated by molecular biologic studies in which the GABA binding site is eliminated from the channel protein but pentobarbital can still activate the channel.¹⁰² Direct radioligand binding studies have demonstrated that benzodiazepines bind to the GABA_A receptor at nanomolar concentrations and that other anesthetics can modulate binding but do not bind directly to the benzodiazepine site.^93,103 A series of more complex studies examining the interactions between barbiturates, anesthetic steroids, and benzodiazepines indicates that these three classes of drugs cannot be acting at the same sites.⁹³ The actions of anesthetics on GABA_A receptors are further complicated by the observation that steroid anesthetics can produce different effects on GABA_A receptors in different brain regions.¹⁰⁴ This suggests the possibility that the specific subunit composition of a GABA_A receptor may encode pharmacologic selectivity. This is well illustrated by benzodiazepine sensitivity, which requires the presence of the γ2 subunit subtype.¹⁰⁵ Similarly, sensitivity to etomidate has been shown to require the presence of a β2 or β3 subunit.¹⁰⁶ More recently, it has been shown that the presence of a δ or ε subunit in a GABA_A receptor confers insensitivity to the potentiating effects of some anesthetics.^107,108

Interestingly, GABA_A receptors composed of ρ-type subunits (referred to as GABA_C receptors) have been shown to be inhibited rather than potentiated by volatile anesthetics.¹⁰⁹ This property has been exploited, using molecular biologic techniques, by constructing chimeric receptors composed of part of the ρ-receptor coupled to part of an α, β, or glycine receptor subunit. By screening these chimeras for anesthetic sensitivity, regions of the α, β, and glycine subunits responsible for anesthetic sensitivity have been identified. Based on the results of these chimeric studies, site-directed mutagenesis studies were performed to identify the specific amino acids responsible for conferring anesthetic sensitivity. These studies revealed two critical amino acids, near the extracellular regions of transmembrane domains 2 and 3 (TM2, TM3) of the glycine and GABA_A receptors that are required for volatile anesthetic potentiation of agonist effect.¹¹⁰ It is not yet clear if these amino acids represent a volatile anesthetic binding site, or whether they are sites critical to transducing anesthetic-induced conformational changes in the receptor molecule. Interestingly, one of the amino acids shown to be critical to volatile anesthetic effect (TM3 site) has also been shown to be required (in the β₂/β₃ subunit) for the potentiating effects of etomidate.¹¹¹ In contrast, the TM2 and TM3 sites do not appear to be required for the actions of propofol, barbiturates, or neurosteroids.¹¹² Interestingly, a distinct amino acid in the TM3 region of the β₁ subunit of the GABA_A receptor has been shown to selectively modulate the ability of propofol to potentiate GABA agonist effects.¹¹² Recent evidence also indicates that neurosteroids actions on GABA_A receptors occur via interactions with specific sites within the transmembrane spanning regions of the α₁ and β₂ subunits that are distinct from those with which benzodiazepines and pentobarbital act.¹¹³ Collectively, these molecular biologic data provide strong evidence that there are multiple unique binding sites for anesthetics on the GABA_A receptor protein.

Other Ligand-Activated Ion Channels

Other members of the ligand-gated receptor superfamily include the nicotinic acetylcholine receptors (muscle and neuronal types), glycine receptors, and 5-HT₃ receptors. A large body of work has gone into examining the effects of anesthetics on nicotinic acetylcholine receptors. The muscle type of nicotinic receptor has been shown to be inhibited by anesthetic concentrations in the clinical range¹¹⁴ and to be desensitized by higher concentrations of anesthetics.¹¹⁵ The muscle nicotinic receptor is an informative model to study because of its abundance and the wealth of knowledge about its structure. It is, however, not expressed in the CNS and hence not involved in the mechanism of anesthesia. However, a neuronal type of nicotinic receptor, which is widely expressed in the nervous system, might plausibly be involved in anesthetic mechanisms. Older studies looking at neuronal nicotinic receptors in molluscan neurons¹¹⁶ and in bovine chromaffin cells¹¹⁷ indicate that these channels are inhibited by clinical concentrations of volatile anesthetics. More recent studies using cloned and expressed neuronal nicotinic receptor subunits have shown a high degree of subunit and anesthetic selectivity. Acetylcholine-elicited currents are inhibited, in receptors composed of various combinations of α₂, α₄, β₂, and β₄ subunits, by subanesthetic concentrations of halothane¹¹⁸ or isoflurane.¹¹⁹ In contrast, these receptors are relatively insensitive to propofol. Most interestingly, receptors composed of α₇ subunits are completely insensitive to both isoflurane and propofol.^119,120 Subsequent pharmacologic experiments using selective inhibitors of neuronal nicotinic receptors led to the conclusion that these receptors are unlikely to have a major role in immobilization by volatile anesthetics.^121,122 However, they might play a role in the amnestic or hypnotic effects of volatile anesthetics.¹²³

Glycine is an important inhibitory neurotransmitter, particularly in the spinal cord and brainstem. The glycine receptor is a member of the ligand-activated channel superfamily that, like the GABA_A receptor, is a chloride-selective ion channel. A large number of studies have shown that clinical concentrations of volatile anesthetics potentiate glycine-activated currents in intact neurons⁸⁰ and in cloned glycine receptors expressed in oocytes.^124,125 The volatile anesthetics appear to produce their potentiating effect by increasing the affinity of the receptor for glycine.¹²⁵ Propofol,⁹⁶ alphaxalone, and pentobarbital also potentiate glycine-activated currents, whereas etomidate and ketamine do not.¹²⁴ Potentiation of glycine receptor function may contribute to the anesthetic action of volatile anesthetics and some parenteral anesthetics. The 5-HT₃ receptors are also members of the genetically related superfamily of ligand-gated receptor channels. Clinical concentrations of volatile anesthetics potentiate currents activated by 5-hydroxytryptamine in intact cells¹²⁶ and in cloned receptors expressed in oocytes.¹²⁷ In contrast, thiopental inhibits 5-HT₃ receptor currents¹²⁶ and propofol is without effect on these receptor channels.¹²⁷ The 5-HT₃ receptors may play some role in the anesthetic state produced by volatile anesthetics and may also contribute to some unpleasant anesthetic side effects such as nausea and vomiting.

Summary

Several ligand-gated ion channels are modulated by clinical concentrations of anesthetics. Ketamine, N₂O, and xenon inhibit NMDA-type glutamate receptors, and this effect may play a major role in their mechanism of action. A large body of evidence shows that clinical concentrations of many anesthetics potentiate GABA-activated currents in the CNS. This suggests that GABA_A receptors are a probable molecular target of anesthetics. Other members of the ligand-activated ion channel family, including glycine receptors, neuronal nicotinic receptors,

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and 5-HT₃ receptors, are also affected by clinical concentrations of anesthetics and remain plausible anesthetic targets.

Anesthetic Effects on Background Potassium Ion Channels

Certain potassium channels called background or leak channels are activated by both volatile and gaseous anesthetics.¹²⁸ Background or leak channels are so named because they tend to be open at all voltages including the resting membrane potential of neurons, producing a “leak current.” Leak currents can significantly regulate the excitability of neurons in which they are expressed. Anesthetic activation of a leak channel was first observed in a ganglion of the pond snail, Lymnea stagnalis. ¹²⁹ Clinical concentrations of halothane activated this channel called I_K(AN), resulting in silencing of the spontaneous bursting of these neurons (Fig. 5-6A). A similar anesthetic-activated background potassium channel was subsequently found by Winegar and Yost¹³⁰ in the marine mollusk Aplysia. The importance of volatile anesthetic activation of these invertebrate potassium channels has now become apparent with the discovery of a large family of background potassium channels in mammals. These mammalian potassium channels have a unique structure with two pore-forming domains in tandem plus four transmembrane segments (2P/4TM; Fig. 5-6C).¹³¹ Patel et al¹³² have studied the effects of volatile anesthetics on several members of the mammalian 2P/4TM family. They have shown that TREK-1 channels are activated by clinical concentrations of chloroform, diethyl ether, halothane, and isoflurane (Fig. 5-6B). In contrast, closely related TRAAK channels are insensitive to all the volatile anesthetics, and TASK channels are activated by halothane and isoflurane, inhibited by diethyl ether, and unaffected by chloroform. These authors went on to show that the C-terminal regions of TASK and TREK-1 contain amino acids essential for anesthetic action.¹³² More recently, TREK-1 but not TASK was found to be activated by clinical concentrations of the gaseous anesthetics: xenon, nitrous oxide, and cyclopropane.¹³³ Thus, activation of background K⁺ channels in mammalian vertebrates could be an important and general mechanism through which inhalational and gaseous anesthetics regulate neuronal resting membrane potential and thereby excitability. Indeed, genetic evidence argues for a role of these channels in producing anesthesia (see later discussion).

Figure 5-6. Volatile anesthetics activate background K+ channels. A: Halothane reversibly hyperpolarizes a pacemaker neuron from Lymnaea stagnalis (the pond snail) by activating IKan. B: Halothane (300 µM) activates human recombinant TREK-1 channels expressed in COS cells. The figure shows current–voltage relationships with reversal potential (Vrev) of -88 mV, indicative of a K+ channel. C: Predicted structure of a typical subunit of the mammalian background K+ channels. Note the four transmembrane spanning segments (in black) and the two pore-forming domains (P1 and P2). Some but not all of these 2P/4TM K+ channels are activated by volatile anesthetics. D: Phylogenetic tree for the 2P/4TM family. (Reproduced with permission from Franks NP, Lieb WR: Background K+ channels: An important target for anesthetics? Nat Neurosci 2:395, 1999.)

Summary

Recent evidence suggests that members of the 2P/4TM family of background potassium channels may be important in producing some components of the anesthetic state.

What is the Chemical Nature of Anesthetic Target Sites?

The Meyer-Overton Rule

More than 100 years ago, Meyer¹³⁴ and Overton¹³⁵ independently observed that the potency of gases as anesthetics was strongly correlated with their solubility in olive oil (Fig. 5-7).

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This observation has significantly influenced thinking about anesthetic mechanisms in two ways. First, since a wide variety of structurally unrelated compounds obey the Meyer-Overton rule, it has been reasoned that all anesthetics are likely to act at the same molecular site. This idea is referred to as the Unitary Theory of Anesthesia. Second, it has been argued that since solubility in a specific solvent strongly correlates with anesthetic potency, the solvent showing the strongest correlation between anesthetic solubility and potency is likely to most closely mimic the chemical and physical properties of the anesthetic target site in the CNS. Based on this reasoning, the anesthetic target site was assumed to be hydrophobic in nature.

Figure 5-7. The Meyer-Overton rule. There is a linear relationship (on a log–log scale) between the oil/gas partition coefficient and the anesthetic potency (minimum alveolar concentration, MAC) of a number of gases. The correlation between lipid solubility and MAC extends over a 70,000-fold difference in anesthetic potency. (Reproduced with permission from Tanfiuji Y, Eger EI, Terrell RC: Some characteristics of an exceptionally potent inhaled anesthetic: thiomethoxyflurane. Anesth Analg 56:387, 1977.)

The Meyer-Overton correlation suffers from two limitations: (1) it only applies to gases and volatile liquids since olive oil/gas partition coefficients cannot be determined for liquid anesthetics, and (2) olive oil is a poorly characterized mixture of oils. To circumvent these limitations, attempts have been made to correlate anesthetic potency with water/solvent partition coefficients. To date, the octanol/water partition coefficient best correlates with anesthetic potency. This correlation holds for a variety of classes of anesthetics and spans a 10,000-fold range of anesthetic potencies.¹³⁶ The properties of the solvent octanol suggest that the anesthetic site is likely to be amphipathic, having both polar and nonpolar characteristics.

Exceptions to the Meyer-Overton Rule

Halogenated compounds exist that are structurally similar to the inhaled anesthetics yet are convulsants rather than anesthetics.¹³⁷ There are also convulsant barbiturates¹³⁸ and neurosteroids.¹³⁹ One convulsant compound, fluorothyl (hexafluorodiethyl ether) has been shown to cause seizures in 50% of mice at 0.12 vol%, but to produce anesthesia at higher concentrations (EC₅₀ = 1.22 vol%).¹⁴⁰ The concentration of fluorothyl required to produce anesthesia is approximately predicted by the Meyer-Overton rule. In contrast, several polyhalogenated alkanes have been identified that are convulsants, but that do not produce anesthesia. Based on the olive oil/gas partition coefficients of these compounds, anesthesia should have been achieved within the range of concentrations studied.¹⁴¹ The end point used to determine the anesthetic effect of these compounds was movement in response to a noxious stimulus (MAC). Interestingly, some of these polyhalogenated compounds do produce amnesia in animals.¹⁴² These compounds are thus referred to asnonimmobilizers rather than as nonanesthetics. Several polyhalogenated alkanes have also been identified that anesthetize mice, but only at concentrations 10 times those predicted by their oil/gas partition coefficients¹⁴¹; these compounds are referred to as transitional compounds. The nonimmobilizers and transitional compounds have been proposed as a “litmus test” for the relevance of anesthetic effects observed in vitro to those observed in the whole animal.

In several homologous series of anesthetics, anesthetic potency increases with increasing chain length until a certain critical chain length is reached. Beyond this critical chain length, compounds are unable to produce anesthesia, even at the highest attainable concentrations. In the series of n-alkanols, for example, anesthetic potency increases from methanol through dodecanol; all longer alkanols are unable to produce anesthesia.¹⁴³ This phenomenon is referred to as the cutoff effect. Cutoff effects have been described for several homologous series of anesthetics including n-alkanes, n-alkanols, cycloalkanemethanols,¹⁴⁴ and perfluoroalkanes.¹⁴⁵ While the anesthetic potency in each of these homologous series of anesthetics shows a cutoff, a corresponding cutoff in octanol/water or oil/gas partition coefficients has not been demonstrated. Therefore, compounds above the cutoff represent a deviation from the Meyer-Overton rule.

A final deviation from the Meyer-Overton rule is the observation that enantiomers of anesthetics differ in their potency as anesthetics. Enantiomers (mirror-image compounds) are a class of stereoisomers that have identical physical properties, including identical solubility in solvents such as octanol or olive oil. Animal studies with the enantiomers of barbiturate anesthetics,^146,147 ketamine,⁸⁹ neurosteroids,⁹⁷ etomidate,¹⁴⁸ and isoflurane¹⁴⁹ all show enantioselective differences in anesthetic potency. These differences in potency range in magnitude from a more than tenfold difference between the enantiomers of etomidate or the neurosteroids to a 60% difference between the enantiomers of isoflurane. It is argued that a major difference in anesthetic potency between a pair of enantiomers could only be explained by a protein-binding site (see “Protein Theories of Anesthesia”); this appears to be the case for etomidate and the neurosteroids. Enantiomeric pairs of anesthetics have also been used to study anesthetic actions on ion channels. It is argued that if an anesthetic effect on an ion channel contributes to the anesthetic state, the effect on the ion channel should show the same enantioselectivity as is observed in whole-animal anesthetic potency. Early studies showed that the (+)-isomer of isoflurane is 1.5 to 2 times more potent than the (-)-isomer in eliciting an anesthetic-activated potassium current, in potentiating GABA_Acurrents, and in inhibiting the current mediated by a neuronal nicotinic acetylcholine receptor.^99,116 In contrast, the stereoisomers of isoflurane are equipotent in their effects on a voltage-activated potassium current and in their effects on lipid phase-transition temperature.¹¹⁶ Studies with the neurosteroids⁹⁷ and etomidate¹⁴⁸ show that these anesthetics exert enantioselective effects on GABA_A currents that parallel the enantioselective effects observed for anesthetic potency.

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The exceptions to the Meyer-Overton rule do not obviate the importance of the rule. They do, however, indicate that the properties of a solvent such as octanol describe some, but not all, of the properties of an anesthetic binding site. Compounds that deviate from the Meyer-Overton rule suggest that anesthetic target site(s) are also defined by other properties including size and shape.

In defining the molecular target(s) of anesthetic molecules one must be able to account both for the Meyer-Overton rule and for the well-defined exceptions to this rule. It has sometimes been suggested that a correct molecular mechanism of anesthesia should also be able to account for pressure reversal. Pressure reversal is a phenomenon whereby the concentration of a given anesthetic needed to produce anesthesia is greatly increased if the anesthetic is administered to an animal under hyperbaric conditions. The idea that pressure reversal is a useful tool for elucidating mechanisms of anesthesia is based on the assumption that pressure reverses the specific physicochemical actions of the anesthetic that are responsible for producing anesthesia; that is to say, pressure and anesthetics act on the same molecular targets. However, recent evidence suggests that pressure reverses anesthesia by producing excitation that physiologically counteracts anesthetic depression, rather than by acting as an anesthetic antagonist at the anesthetic site of action.¹⁵⁰Therefore, in the following discussion of molecular targets of anesthesia, pressure reversal will not be further mentioned.

Lipid versus Protein Targets

Anesthetics might interact with several possible molecular targets to produce their effects on the function of ion channels and other proteins. Anesthetics might dissolve in the lipidbilayer, causing physicochemical changes in membrane structure that alter the ability of embedded membrane proteins to undergo conformational changes important for their function. Alternatively, anesthetics could bind directly to proteins (either ion channel proteins or modulatory proteins), thus either (1) interfering with binding of a ligand (e.g., a neurotransmitter, a substrate, a second messenger molecule) or (2) altering the ability of the protein to undergo conformational changes important for its function. The following section summarizes the arguments for and against lipid theories and protein theories of anesthesia.

Lipid Theories of Anesthesia

The elucidation of the Meyer-Overton rule suggested that anesthetics interact with a hydrophobic target. To investigators in the early part of the 20th century, the most logical hydrophobic target was a lipid. In its simplest incarnation, the lipid theory of anesthesia postulates that anesthetics dissolve in the lipid bilayers of biological membranes and produce anesthesia when they reach a critical concentration in the membrane. Consistent with this hypothesis, the membrane/gas partition coefficients of anesthetic gases in pure lipid bilayers correlate strongly with anesthetic potency.¹⁵¹ Also, consistent with the lipid theories, various membrane perturbations are produced by general anesthetics; however, the magnitude of these changes produced by clinical concentrations of anesthetics are quite small and are thought to be very unlikely to disrupt nervous system function.¹⁵² While some of the more sophisticated lipid theories can account for the cutoff effect and impotence of nonimmobilizers, no lipid theory can plausibly explain all anesthetic pharmacology. Thus, most investigators do not consider membranes/lipids as the most likely target of general anesthetics.

Protein Theories of Anesthesia

The Meyer-Overton rule could also be explained by the direct interaction of anesthetics with hydrophobic sites on proteins. Three types of hydrophobic sites on proteins might interact with anesthetics:

1. Hydrophobic amino acids comprise the core of water-soluble proteins. Anesthetics could bind in hydrophobic pockets that are fortuitously present in the protein core.

2. Hydrophobic amino acids also form the lining of binding sites for hydrophobic ligands. For example, there are hydrophobic pockets in which fatty acids tightly bind on proteins such as albumin and the low-molecular-weight fatty acid–binding proteins. Anesthetics could compete with endogenous ligands for binding to such sites on either water-soluble or membrane proteins.

3. Hydrophobic amino acids are major constituents of the α-helices, which form the membrane-spanning regions of membrane proteins; hydrophobic amino acid side chains form the protein surface that faces the membrane lipid. Anesthetic molecules could interact with the hydrophobic surface of these membrane proteins, disrupting normal lipid–protein interactions and possibly directly affecting protein conformation. This last possibility would involve the interaction of many anesthetic molecules with each membrane protein molecule and would probably be a nonselective interaction between anesthetic molecules and all membrane proteins.

Direct interactions of anesthetic molecules with proteins would not only satisfy the Meyer-Overton rule, but would also provide the simplest explanation for compounds that deviate from this rule. Any protein-binding site is likely to be defined by properties such as size and shape in addition to its solvent properties. Limitations in size and shape could reduce the binding affinity of compounds beyond the cutoff, thus explaining their lack of anesthetic effect. Enantioselectivity is also most easily explained by a direct binding of anesthetic molecules to defined sites on proteins; a protein-binding site of defined dimensions could readily distinguish between enantiomers on the basis of their different shape. Protein-binding sites for anesthetics could also explain the convulsant effects of some polyhalogenated alkanes. Different compounds binding (in slightly different ways) to the same binding pocket can produce different effects on protein conformation and hence on protein function. For example, there are three kinds of compounds that can bind at the benzodiazepine binding site on the GABA_A channel: agonists, which potentiate GABA effects and produce sedation and anxiolysis; inverse agonists, which promote channel closure and produce convulsant effects; and antagonists, which produce no effect on their own but can competitively block the effects of agonists and inverse agonists. By analogy, polyhalogenated alkanes could be inverse agonists, binding at the same protein sites at which halogenated alkane anesthetics are agonists. The evidence for direct interactions between anesthetics and proteins is briefly reviewed in the following section.

Evidence for Anesthetic Binding to Proteins

A breakthrough in protein theories of anesthesia was the demonstration that a purified water-soluble protein, firefly luciferase, could be inhibited by general anesthetics. This provided the important proof-of-principle that anesthetics could bind to proteins in the absence of membranes. Numerous studies have extensively characterized anesthetic inhibition of

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firefly luciferase activity and have revealed the following^153,154:

1. Anesthetics inhibit firefly luciferase activity at concentrations very similar to those required to produce clinical anesthesia.

2. The potency of anesthetics as inhibitors of firefly luciferase activity correlates strongly with their potency as anesthetics, in keeping with the Meyer-Overton rule.

3. Halothane inhibition of luciferase activity is competitive with respect to the substrate D-luciferin.

4. Inhibition of firefly luciferase activity shows a cutoff in anesthetic potency for both n-alkanes and n-alkanols.

Based on these studies it can be inferred that a wide variety of anesthetics can bind in the luciferin-binding pocket of firefly luciferase. The fact that anesthetic inhibition of luciferase activity is consistent with the Meyer-Overton rule, occurs at clinical anesthetic concentrations, and explains the cutoff effect suggests that the luciferin-binding pocket may have physical and chemical characteristics similar to those of a putative anesthetic binding site in the CNS.

More direct approaches to study anesthetic binding to proteins have included NMR spectroscopy and photoaffinity labeling. Based on early studies by Wishnia and Pinder,^155,156 it was suspected that anesthetics could bind to several fatty acid–binding proteins, including β-lactoglobulin and bovine serum albumin (BSA). ¹⁹F-NMR spectroscopic studies confirmed¹⁵⁷this, and demonstrated that isoflurane binds to approximately three saturable binding sites on BSA. Isoflurane binding is eliminated by coincubation with oleic acid, suggesting that isoflurane binds to the fatty acid–binding sites on albumin. Other anesthetics, including halothane, methoxyflurane, sevoflurane, and octanol, compete with isoflurane for binding to BSA.¹⁵⁸ The studies with BSA provide direct evidence that a variety of anesthetics can compete for binding to the same site on a protein. Using this BSA model, it was subsequently shown that anesthetic binding sites could be identified and characterized using a photoaffinity labeling technique. The anesthetic halothane contains a carbon–bromine bond. This bond can be broken by ultraviolet light generating a free radical. That free radical allows the anesthetic to permanently (covalently) label the anesthetic binding site. Eckenhoff and Shuman¹⁵⁹ used ¹⁴C-labeled halothane to photoaffinity-label anesthetic binding sites on BSA, and obtained results virtually identical to those obtained using NMR spectroscopy. Eckenhoff¹⁶⁰ subsequently has identified the specific amino acids that are photoaffinity-labeled by [¹⁴C]halothane. NMR and photoaffinity-labeling techniques have also been applied to several other proteins. For example, saturable binding of halothane to the luciferin-binding site on firefly luciferase has been directly confirmed using NMR and photoaffinity-labeling techniques.¹⁶¹ Most recently, Husain and colleagues¹⁶² have developed a general anesthetic that is an analog of octanol and functions as a photoaffinity label. This compound, 3-diazyrinyloctanol, binds to specific sites on the nicotinic acetylcholine receptor.

Although NMR and photoaffinity techniques can provide extensive information about anesthetic binding sites on proteins, they cannot reveal the details of the three-dimensional structure of these sites. X-Ray diffraction crystallography can provide this kind of three-dimensional detail and has been used to study anesthetic interactions with a small number of proteins. To date, it has been difficult to crystallize membrane proteins; thus, these studies have been limited to water-soluble proteins. Firefly luciferase has been crystallized in the presence and absence of the anesthetic bromoform. X-Ray diffraction studies of these crystals showed that the anesthetic does bind in the luciferin-binding pocket, as had been inferred from functional studies. Interestingly, two molecules of bromoform bind in the luciferin pocket—one that is likely to compete directly with luciferin for binding and one that is not.¹⁶³ The binding data with firefly luciferase is of particular interest because it demonstrates that anesthetics can bind to endogenous ligand binding sites and that this binding strongly correlates with anesthetic inhibition of protein function. The same group has also crystallized human serum albumin in the presence of either propofol or halothane. The x-ray crystallographic data demonstrate binding of both anesthetics to preformed pockets that had been shown previously to bind fatty acids.¹⁶⁴ Given that both of these anesthetics bind to serum albumin at clinical concentrations, these data give the best insight yet into the structure of an anesthetic binding pocket.

A recent approach to study anesthetic interactions with proteins has been to employ site-directed mutagenesis of candidate anesthetic targets, coupled with molecular modeling to make predictions about the location and structure of anesthetic binding sites. For example, Wick and colleagues¹⁶⁵ have used this approach to predict the location and structure of the alcohol binding site on GABA_A and glycine receptors. Similarly, the likely neurosteroid binding sites for activation and potentiation of the GABA_A receptor were found by extensive site-directed mutagenesis experiments.¹¹³ A related approach has been to develop model proteins to define the structural requirements for an anesthetic binding site. Using this approach, Johansson et al¹⁶⁶ have shown that a four–α-helix bundle with a hydrophobic core can bind volatile anesthetics at concentrations (K_D) similar to those required to produce anesthesia.¹⁶⁶

Summary

Unequivocal evidence from studies using water-soluble proteins demonstrates that anesthetics can bind to hydrophobic pockets on proteins. Functional and binding studies with firefly luciferase demonstrate that anesthetics can bind to a protein site at clinically relevant concentrations in a manner that can account for the Meyer-Overton rule and deviations from it. Evidence that direct anesthetic–protein binding interactions may be responsible for anesthetic effects on ion channels in the CNS remains indirect; stereoselectivity currently offers the strongest indirect argument.

Overall, current evidence strongly indicates protein rather than lipid as the molecular target for anesthetic action. While the long-standing controversy between lipid and protein theories of anesthesia may be behind us, numerous unanswered questions remain about the details of anesthetic–protein interactions, including:

1. What is the stoichiometry of anesthetic binding to a protein (i.e., Do many anesthetic molecules interact with a single protein molecule or only a few)?

2. Do anesthetics compete with endogenous ligands for binding to hydrophobic pockets on protein targets or do they bind to fortuitous cavities in the protein?

3. Do all anesthetics bind to the same pocket on a protein or are there multiple hydrophobic pockets for different anesthetics?

4. How many proteins have hydrophobic pockets in which anesthetics can bind at clinically used concentrations?

How Are the Molecular Effects of Anesthetics Linked to Anesthesia in the Intact Organism?

The previous sections have described how anesthetics affect the function of a number of ion channels and signaling proteins, probably via direct anesthetic–protein interactions. It is

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unclear which, if any, of these effects of anesthetics on protein function are necessary and/or sufficient to produce anesthesia in an intact organism. A number of approaches have been employed to try to link anesthetic effects observed at a molecular level to anesthesia in intact animals. These approaches and their pitfalls are briefly explored in the following section.

Pharmacologic Approaches

An experimental paradigm frequently used to study anesthetic mechanisms is to administer a drug thought to act specifically at a putative anesthetic target (e.g., a receptor agonist or antagonist, an ion channel activator or antagonist), then determine whether the drug has either increased or decreased the animal's sensitivity to a given anesthetic. The underlying assumption is that if a change in anesthetic sensitivity is observed, then the anesthetic is likely to act via an action on the specific target of the administered drug. This is a largely flawed strategy that has nonetheless produced a huge literature. The drugs used to modulate anesthetic sensitivity usually have their own direct effects on CNS excitability and thus indirectly affect anesthetic requirements. For example, while α₂-adrenergic agonists decrease halothane MAC,¹⁶⁷ they are profound CNS depressants in their own right and produce anesthesia by mechanisms distinct from those used by volatile anesthetics. Thus, the “MAC-sparing” effects of α₂-agonists provide little insight into how halothane works. A more useful pharmacologic strategy would be to identify drugs that have no effect on CNS excitability but prevent the effects of given anesthetics. Currently, however, there are no such anesthetic antagonists. Development of specific antagonists for anesthetic agents would provide a major tool for linking anesthetic effects at the molecular level to anesthesia in the intact organism, and might also be of significant clinical utility.

An alternative pharmacologic approach is to develop “litmus tests” for the relevance of anesthetic effects observed in vitro. One such test takes advantage of compounds that are nonanesthetic despite the predictions of the Meyer-Overton rule. It is argued that “a site affected by these nonanesthetic compounds is unlikely to be relevant to the production of anesthesia.”¹⁴¹ A similar argument uses stereoselectivity as the discriminator and argues that a site that does not show the same stereoselectivity as that observed for whole animal anesthesia is unlikely to be relevant to the production of anesthesia.¹⁶⁸ Although these tests may be useful, they are very dependent on the assumption that anesthesia is produced via drug action at a single site. For example, a nonanesthetic might depress CNS excitability via its actions on an important anesthetic target site while simultaneously producing counterbalancing excitatory effects at a second site. In this case the “litmus test” would incorrectly eliminate the anesthetic site as irrelevant to whole-animal anesthesia. This example is quite plausible given the convulsant effects of many of the nonanesthetic polyhalogenated hydrocarbons. Another sort of litmus test is to selectively antagonize the putative anesthetic target so that this target is no longer functional. If anesthetic effects are mediated through this target, inactivation of the target by the antagonist should result in anesthetic resistance. Using this logic, the modest MAC-sparing effects of GABA_A and glycine receptor antagonists were used to argue that both GABA_A and glycine receptors mediate some but not all of the immobilizing effects of volatile anesthetics in rodents.^169,170 This same group used the lack of effect of neuronal nicotinic antagonists on isoflurane MAC to conclude that these receptors had no role in volatile anesthetic immobilization.¹²² As with many pharmacologic results, the issues of specificity and efficacy of the antagonists prevent these experiments from being definitive. Nevertheless, these results are consistent with the findings that volatile anesthetics affect the function of a large number of important neuronal proteins and no one target is likely to mediate all of the effects of these drugs.

Genetic Approaches

An alternative approach to study the relationship between anesthetic effects observed in vitro and whole-animal anesthesia is to alter the structure or abundance of putative anesthetic targets and determine how this affects whole-animal anesthetic sensitivity. Genetic techniques provide the most reliable and versatile methods for changing the structure or abundance of putative anesthetic targets. The first true genetic screen for mutants with altered general anesthetic sensitivity was performed in the nematode C. elegans by Phil Morgan and Margaret Sedensky.¹⁷¹ They screened for altered sensitivity to supraclinical concentrations of halothane. High halothane concentrations were used because they are required to immobilize C. elegans. The first mutant isolated had a threefold reduction in its EC₅₀ for halothane. The mutation was genetically mapped and found to be a loss-of-function allele of the unc-79 gene, which encodes a large neuronal protein very similar in sequence to a human protein.¹⁷² The cellular function of either the C. elegans or human protein is unknown. In the absence of anesthetics, unc-79 mutants have an interesting locomotion defect called fainting. Normal C. elegans worms crawl almost continuously whereasunc-79 mutants appear to faint where they spontaneously stop moving for extended periods of time. In testing other such mutants, Humphrey et al¹⁷² and Morgan and Sedensky¹⁷³found that, in general, “fainters” were hypersensitive to halothane. Subsequent extensive genetic screens and mapping of fainting mutants have led to a focus on a novel presumptive cation channel, NCA-1/NCA-2, that controls halothane sensitivity in both C. elegans and in the fruit fly Drosophila.¹⁷² This remarkable conservation of the anesthetic hypersensitivity phenotype across such divergent species argues for a fundamental role of NCA-1/NCA-2 in the action of halothane.

Clinical concentrations of volatile anesthetics do not immobilize C. elegans, but they do produce behavioral effects including loss of coordinated movement.¹⁷⁴ Crowder and colleagues¹⁷⁴ have screened for mutants that are resistant to anesthetic-induced uncoordination and found that mutations in a set of genes encoding proteins regulating neurotransmitter release control anesthetic sensitivity. The gene with the largest effect encoded syntaxin 1A, a neuronal protein highly conserved from C. elegans to humans and essential for fusion of neurotransmitter vesicles with the presynaptic membrane.¹⁷⁵ Importantly, some syntaxin mutations produced hypersensitivity to volatile anesthetics while others conferred resistance. These allelic differences in anesthetic sensitivity could not be accounted for by effects on the process of transmitter release itself^56,175; rather, the genetic data argued that syntaxin interacts with a protein critical for volatile anesthetic action, perhaps an anesthetic target. Recently, a highly evolutionarily conserved presynaptic protein called UNC-13 in C. elegans was implicated in this presynaptic volatile anesthetic mechanism.¹⁷⁶ UNC-13 is required for normal isoflurane sensitivity, unc-13 mutants are fully resistant to the effects of clinical concentration of isoflurane, and isoflurane prevents the normal synaptic localization of UNC-13. Whether UNC-13 is a direct target of volatile anesthetics is unknown. This same laboratory has also shown by mutant analysis that an NMDA glutamate receptor subunit is essential for nitrous oxide sensitivity in C. elegans¹⁷⁷ and that another glutamate receptor subunit is required for the effects of Xenon.¹⁷⁸

In Drosophila, clinical concentrations of volatile anesthetics disrupt negative geotaxis behavior and response to a noxious light

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or heat stimulus.^179,180,181 Using one or more of these anesthetics effects, Krishnan and Nash¹⁷⁹ performed a forward genetic screen for halothane resistance. The results of this screen have led to a focus on the Drosophila homolog of nca-1/2. As previously discussed, mutants in the Drosophila homolog of nca-1/2 are hypersensitive to halothane like the C. elegansmutants.¹⁷² The synergy of both Drosophila and C. elegans genetics should lead to an understanding of how this channel controls volatile anesthetic sensitivity.

In mammals, the most powerful genetic model organism is mouse, where techniques have been developed to alter or delete any gene of interest. The GABA_A receptor has been extensively studied using mouse genetic techniques.^182,183 The genes encoding for various subunits of the GABA_A receptor have been mutated so that they are either nonfunctional (gene knockouts) or so that they have altered amino acids that might produce altered function (gene knockins). Knockouts of three α subunits of the GABA_A receptor have been tested for their anesthetic sensitivity. Deletion of the α1 subunit does not alter sensitivity of mice to the hypnotic effects of pentobarbital.¹⁸⁴ Similarly, α6 subunit knockout mice are normally sensitive to halothane and enflurane.¹⁸⁵ However, α5 knockout mice are resistant to learning impairment by etomidate.¹⁸⁶ Knockin mouse strains have been generated for several of the α-subunits, primarily for examining benzodiazepine action. The loss of various aspects of benzodiazepine action in these strains demonstrated that the α1 subunit mediates the sedative and amnestic actions, and is partially required for its anticonvulsant properties. Similarly, the α2 subunit has been shown to be essential for anxiolysis by diazepam, and α3 and α5 knockin strains are partially resistant to its myorelaxant effects. Finally, a mouse expressing a double mutated α1 subunit, α1(S270H, L277A), has recently been tested for its anesthetic sensitivity.^187,188 The α1S270H mutation has been shown to block GABA potentiation by volatile anesthetics, but the mutation also increases native sensitivity to GABA, confounding interpretation of the data. Moreover, α1S270H single-mutant mice are quite abnormal behaviorally and are prone to anesthetic-induced seizure activity.¹⁸⁹ Thus, a second mutation, L277A, was introduced into the α1 subunit that compensated for the change in native gating properties. The α1(S270H, L277A) mice are viable and behaviorally normal. These mice are mildly resistant to the ataxic effects of isoflurane and enflurane; however, the potency of the drugs in MAC and fear-conditioning assays (a measure of learning) are not altered by the double-mutant α1 subunit.

In vitro electrophysiological experiments show that a specific β3 subunit point mutation, β3(N265M), blocks the action of etomidate and propofol on the GABA_A receptor without greatly altering receptor function in the absence of drug.^111,190 A β3(N265M) knockin strain was generated and found to be insensitive to the immobilizing effects of etomidate, propofol, and pentobarbital.^191,192 However, the β3(N265M) mice are not completely resistant to the loss-of-righting reflex by these anesthetics, indicating that other targets mediate this behavioral effect. Interestingly, the respiratory depressant effects of etomidate and propofol are also blocked by the β3(N265M) mutation, but the cardiovascular and hypothermic actions of the drugs are not.¹⁹³ The β3(N265M) mice show a slightly reduced sensitivity to the immobilizing actions of volatile anesthetics, suggesting that the β3 subunit may play a minor role in immobilization, but the mutant has unaltered sensitivity to the amnestic effects of isoflurane.¹⁹⁴ A similar approach for the β2 subunit has shown that it is critical for the sedating but not anesthetic action of etomidate.^195,196 Finally, strains carrying a knockout mutation of the δ subunit of the GABA_A receptor have a shorter duration of neurosteroid-induced loss-of-righting reflex whereas their sensitivity to other intravenous and volatile anesthetics is unchanged.¹⁹⁷ Thus, the δ subunit may play a relatively specific role in neurosteroid action.

The roles in anesthetic sensitivity of two of the background potassium channels have been tested in limited mouse genetic studies. A TREK-1 knockout mouse was found to be significantly resistant to multiple volatile anesthetics for MAC and loss-of-righting reflex endpoints.¹⁹⁸ The volatile anesthetic resistance of the TREK-1 knockout is substantial, particularly for halothane where MAC was increased by 48%. Importantly, the TREK-1 knockout mice have a normal sensitivity to pentobarbital, indicating specificity for volatile anesthetics consistent with previous electrophysiological data. Recently, Westphalen et al¹⁹⁹ of the Hemmings laboratory has used the TREK-1 knockout strain to test the hypothesis that TREK-1 mediates some of the presynaptic inhibitory effects of volatile anesthetics. Indeed, glutamate release from synaptosomes prepared from the TREK-1 knockout strain is significantly resistant to inhibition by halothane compared to release from wild type control synaptosomes. The role of TASK-2, another two-pore background potassium channel, has been similarly tested by measuring the MAC of a TASK-2 knockout mouse. However, unlike for TREK-1, the TASK-2 knockout has MAC values similar to wild type controls for desflurane, halothane, and isoflurane.⁷⁷ This result is somewhat surprising given that TASK-2 is strongly activated by halothane and isoflurane and may be explained by an overall reduced expression in the nervous system compared to TREK-1.¹²⁸

Summary

Results from both invertebrate and vertebrate genetics indicate that multiple proteins control volatile anesthetic sensitivity. Some of these may be anesthetic targets and some not. Certain GABA_A receptor subunits and the TREK-1 background potassium channel are very likely to be targets relevant to general anesthesia, but are probably not the only ones. The mammalian electrophysiological data and the genetic evidence in C. elegans both implicate the NMDA glutamate receptor as the primary target of nitrous oxide. Similarly, elegant electrophysiological and genetic experiments have shown that the GABA_A receptor is the primary mediator for immobilization by etomidate, propofol, and pentobarbital.

Conclusions

In this chapter evidence has been reviewed concerning the anatomic, physiologic, and molecular loci of anesthetic action. It is clear that all anesthetic actions cannot be localized to a specific anatomic site in the CNS; indeed, some evidence suggests that different components of the anesthetic state may be mediated by actions at disparate anatomic sites. The actions of anesthetics also cannot be localized to a specific physiologic process. While there is consensus that anesthetics ultimately affect synaptic function as opposed to intrinsic neuronal excitability, the effects of anesthetics depend on the agent and synapse studied and can affect presynaptic and/or postsynaptic function. At a molecular level, volatile anesthetics show some selectivity, but still affect the function of multiple ion channels and synaptic proteins. The intravenous anesthetics, etomidate, propofol, and barbiturates, are more specific with the GABA_A receptor as their major target. Although it is likely that these effects are mediated via direct protein–anesthetic interactions, it appears that there are numerous proteins that can directly interact with anesthetics. Genetic data plainly demonstrate that the unitary theory of anesthesia is not correct. No single mechanism is responsible for the effects of all general anesthetics, nor does a single mechanism account for all of the effects of a single anesthetic, at least where it has been examined. Figure 5-8provides a simple model of the molecular

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and cellular effects of general anesthetics. This cartoon is not meant to include all potential targets of general anesthetics. Rather, only those molecules with strong evidence for importance in anesthetic action from multiple different approaches are shown.

Figure 5-8. A multisite model for anesthesia. Anesthetics are grouped according to similarity of mechanism. Arrows indicate activation or potentiation and “T's” indicate inhibition or antagonism. The neurophysiological effects of general anesthetics are lumped into neuronal excitability (the probability of a neuron firing and propagating an axon potential) and excitatory neurotransmission (synaptic activity at excitatory synapses such as glutamatergic). Neuronal excitability in this context is the sum of both intrinsic and extrinsic factors (e.g., GABAergic inhibition).

Although the precise molecular interactions responsible for producing anesthesia have not been fully elucidated, it has become clear that anesthetics do act via selective effects on specific molecular targets. The technologic revolutions in molecular biology, genetics, and cell physiology make it likely that the next decade will provide some answers to the century-old pharmacologic puzzle of the molecular mechanism of anesthesia.

Acknowledgment

The authors acknowledge generous ongoing funding support from National Institute of General Medical Sciences, Bethesda, Maryland, for ASE-P01 GM047969 and CMC-R01 GM59781.

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Editors: Barash, Paul G.; Cullen, Bruce F.; Stoelting, Robert K.; Cahalan, Michael K.; Stock, M. Christine