ANESTHETIC PHARMACOLOGY INTERNATIONAL SOCIETY FOR ANAESTHETIC PHARMACOLOGY

SECTION EDITOR
JAMES G. BOVILL
REVIEW ARTICLE

Inhaled Anesthetics and Immobility: Mechanisms, Mysteries,

and Minimum Alveolar Anesthetic Concentration
James M. Sonner, MD*, Joseph F. Antognini, MD†, Robert C. Dutton, MD*, Pamela Flood, MD‡,
Andrew T. Gray, MD, PhD*, R. Adron Harris, PhD§, Gregg E. Homanics, PhD储,
Joan Kendig, PhD¶, Beverley Orser, MD#, Douglas E. Raines, MD**, James Trudell, PhD¶,
Bryce Vissel, PhD††, and Edmond I Eger II, MD*
*Department of Anesthesia and Perioperative Care, University of California, San Francisco, California; †Department of
Anesthesiology, University of California, Davis, California; ‡Columbia University, New York, New York; §University of
Texas, Austin, Texas; 储University of Pittsburgh, Pittsburgh, Pennsylvania; ¶Stanford University, Palo Alto, California;
#University of Toronto, Toronto, Canada; **Department of Anaesthesia, Harvard Medical School, Cambridge,
Massachusetts; and ††Garvan Institute of Medical Research, Darlinghurst, Australia

Studies using molecular modeling, genetic engineer-
ing, neurophysiology/pharmacology, and whole ani-
mals have advanced our understanding of where and
how inhaled anesthetics act to produce immobility
(minimum alveolar anesthetic concentration; MAC) by
actions on the spinal cord. Numerous ligand- and
voltage-gated channels might plausibly mediate MAC,
and specific animo acid sites in certain receptors
present likely candidates for mediation. However, in
vivo studies to date suggest that several channels or re-
ceptors may not be mediators (e.g., ␥-aminobutyric acid
A, acetylcholine, potassium, 5-hydroxytryptamine-3,
opioids, and ␣2-adrenergic), whereas other receptors/
channels (e.g., glycine, N-methyl-d-aspartate, and so-
dium) remain credible candidates.
(Anesth Analg 2003;97:718 –40)

A
fter decades of ignorance, we now know how
some anesthetics (e.g., etomidate) act (1). This
knowledge resulted from information revealed
by multiple approaches to studies of anesthetic mech-
anisms, including studies in whole animals using old-
fashioned pharmacologic techniques, in vivo and in
vitro neurophysiological studies, and the application
of genetic engineering. Genetic engineering allows the
manipulation of receptors and their study in vitro. It
allows the creation of animals (usually mice) with
mutated receptors that do not respond to anesthetics
and that, thereby, serve as tools to test the importance
of a given receptor to a particular anesthetic effect.
This review discusses how these approaches have in-
creased understanding of the mechanisms by which a
particular class of anesthetics, the inhaled anesthetics,
acts. The review focuses on the mechanistic bases for
Dr. Eger is a paid consultant to Baxter Healthcare Corp.
This work was supported by National Institute of General Med-
ical Sciences Grant 1P01GM47818.
Accepted for publication May 21, 2003.
Address correspondence and reprint requests to James M. Sonner,
MD, Department of Anesthesia, S-455, University of California, San
Francisco, CA 94143-0464. Address e-mail to sonnerj@anesthesia.
ucsf.edu.
DOI: 10.1213/01.ANE.0000081063.76651.33
one characteristic of inhaled anesthetics: their capacity
to cause immobility. The standard measure of anes-
thetic potency, MAC (the minimum alveolar concen-
tration of an inhaled anesthetic required to suppress
movement in response to noxious stimulation in 50%
of subjects), reflects this capacity (2). MAC is an anes-
thetic 50% effective dose (ED50).
At the turn of the last century, Overton (3) and
Meyer (4) reported that anesthetic potency correlated
with lipophilicity, suggesting the importance of a
lipid-like phase to anesthetic action. For conventional
and many experimental inhaled anesthetics (5–9), the
correlation is remarkable (Fig. 1). Over the ensuing 80
years, a few theories of narcosis evolved from the
focus on lipids, particularly the membrane bilayer, but
none of these theories withstood experimental scru-
tiny. For example, inhaled anesthetics increase mem-
brane disorder (increase fluidity) (10 –14), and this was
proposed as a basis for anesthesia. However, increases
in body temperature also increase this disorder, but
increases in body temperature increase rather than
decrease MAC (15). The discovery of inhaled com-
pounds with potencies that do not correlate with li-
pophilicity called into question the Meyer-Overton
hypothesis: alcohols are more potent than predicted
from their lipophilicity, whereas some compounds are

©2003 by the International Anesthesia Research Society

718 Anesth Analg 2003;97:718–40 0003-2999/03
ANESTH ANALG
2003;97:718 –40
Figure 1. The potencies (minimum alveolar anesthetic concentra-
tion; MAC) of conventional [nitrous oxide (7), halothane (6), isoflu-
rane (6), sevoflurane (6), desflurane (6), and cyclopropane (6)] and
several unconventional [xenon (9), 1A (1-chloro-1,2,2-
trifluorocyclobutane) (5), benzene (8), and hexafluorobenzene (8)]
inhaled anesthetics in rats correlate with their affinity to a lipid
phase, as defined by the oil/gas partition coefficient. A caveat:
although this relationship applies to all conventional inhaled anes-
thetics, several compounds (e.g., nonimmobilizers) do not conform
to the relationship.
less potent (transitional compounds) (5). For example,
ethanol has an oil/gas partition coefficient of 108 (16),
similar to the value of 98 for isoflurane (17). However,
the MAC of isoflurane in rats is 1.5% (18,19), more
than 10 times larger than the MAC of 0.1% for ethanol
(16). At an extreme are nonimmobilizer compounds,
compounds that have no anesthetic effect, alone or in
combination with known anesthetics, despite a li-
pophilicity that predicts an anesthetizing capacity (5).
For example, 1,2-dichlorohexafluorobutane (also
called F6 or 2N) has an oil/gas partition coefficient of
44 (5), similar to the value of 47 found for sevoflurane
(20), which has a MAC of 2.4% (21,22). However, F6 is
not anesthetic at any concentration, including concen-
trations exceeding 4% (5).
The seminal work of Franks and Lieb (23) shifted
attention from lipids to proteins (specifically, ion
channels) as anesthetic targets. Investigations soon
produced a plethora of plausible anesthetic targets, for
the most part ligand- and voltage-gated channels.
Clinical concentrations of inhaled anesthetics could
enhance the effect of inhibitory neurotransmitter-
gated or voltage-gated (notably, potassium) channels
(i.e., increase the sensitivity of the channels to putative
transmitters) or block the effect of excitatory
neurotransmitter-gated or voltage-gated (notably, so-
dium) channels. They also could block the evoked
REVIEW ARTICLE SONNER ET AL. 719
MECHANISMS UNDERLYING MAC
release of neurotransmitters such as glutamate (24 –
26), but not necessarily all neurotransmitters (27). Be-
cause such studies identify candidate receptors, they
are essential for determining the plausibility of spe-
cific receptors as mediators of immobility, but such
identification does not prove relevance.
Antognini and Schwartz (28), Antognini et al. (29),
Borges and Antognini (30), Rampil et al. (31), and
Rampil (32) provided another key finding: that in-
haled anesthetic-induced immobility largely results
from an action on the spinal cord rather than an action
on higher centers. Their results informed many sub-
sequent studies, several of which are described in this
review.
Finally, the increasing capacity to study and manip-
ulate receptors through genetic engineering provides
a powerful tool for studies of anesthetic mechanisms.
Results of such work complement work from tradi-
tional pharmacology. For example, as will be dis-
cussed, the nonimmobilizer F6 blocks acetylcholine
receptors in vitro, and thus the acetylcholine receptor
is an unlikely target for the immobilizing effect of
inhaled anesthetics. Consistent with this finding, in
vivo blockade of acetylcholine receptors does not affect
MAC.
The Spinal Cord as the Primary Site
Mediating Immobilization
Several studies demonstrate that the spinal cord me-
diates most of the ability of inhaled anesthetics to
produce immobility. The observation a decade ago
that immobility during noxious stimulation does not
correlate with electroencephalographic (EEG) activity
prompted the hypothesis that cortical electrical activ-
ity does not control motor responses to noxious stim-
ulation (33). In rats, precollicular decerebration (31)
(Fig. 2) or complete section of the upper thoracic spi-
nal cord (32) minimally affects isoflurane’s capacity to
suppress movement. In vivo electrophysiologic exam-
ination of the spinal cord suggests that anesthetics can
suppress both sensory (34) and motor (35) neuron
activity. In rats (36) and humans (37), suppression of
movement in response to noxious stimulation corre-
lates with suppression of motor neuron excitability (as
determined by recurrent impulses known as F waves).
In goats, MAC for isoflurane delivered to the whole
body is 1.2%, but delivery to only the brain increases
MAC to nearly 3% (28). The separation with halothane
is still greater (29). Control halothane MAC is 0.9%,
but delivery confined to the head requires 3.4% to
abolish movement. Some goats move during EEG si-
lence and move at the largest cerebral halothane con-
centrations tested. These results indicate that inhaled
anesthetics act primarily on the spinal cord to produce
immobility and that only a minor component of this
720 REVIEW ARTICLE SONNER ET AL.
MECHANISMS UNDERLYING MAC
Figure 2. Decerebration does not change the minimum alveolar
anesthetic concentration (MAC) of isoflurane in rats (31). These data
and similar data gathered in Rampil’s laboratory (32) and Antogni-
ni’s laboratory (28,30) demonstrate that the spinal cord mediates
much or most of the capacity of inhaled anesthetics to produce
immobility. That is, effects on the spinal cord determine MAC.
immobility results from cerebral effects. The common
results from different species and experimental meth-
ods increase the compelling nature of these
conclusions.
But where within the cord do inhaled anesthetics act
to produce immobility? One observation suggests a
potential complexity: if coordinated movement occurs
during anesthesia, it often extends beyond the area
stimulated. For example, stimulation of the tail of a rat
provokes movement of the lower and the upper ex-
tremities. This example suggests initial signal process-
ing by sacral-coccygeal components, transmission to
lumbar and cervical components by ascending projec-
tion tracts, and transmission through cervical compo-
nents systems to effect movement. The next section
shows that the specifics of where and how (as well as
not where and not how), including possible putative
receptors, are now partially known.
What Has Been Learned from In Vitro
Spinal Cord Studies?
Because motor neurons integrate all input and signal
muscles to move, anesthetic effects on motor neurons
may be important to the production of immobility.
Other spinal cord components (primary afferent ter-
minals and interneurons) likely also play a role. The
effect of anesthetics on motor neurons has been exam-
ined in the intact isolated neonatal rat spinal cord.
Volatile anesthetics (38,39) and ethanol (40) depress
motor neuron output evoked by dorsal root
stimulation.
Volatile anesthetics enhance the currents through
gamma-aminobutyric acid (GABA)A and glycine in-
hibitory chloride channels that are produced by the
ANESTH ANALG
2003;97:718 –40
application of GABA or glycine. This action reduces
the excitability of neurons and might contribute to
anesthetic depressant actions. If this is the case, then
blockade of these channels should oppose the depres-
sant effects of anesthetics. In intact spinal cord, block-
ade of GABAA and glycine inhibitory chloride chan-
nels does, in fact, decrease the depressant effects of
anesthetics (41). However, whether blockade is only
partial reduction or is complete cannot be determined
experimentally in this preparation because of uncoor-
dinated fluctuations in the baseline brought about by
the convulsant effects of these blocking drugs.
Acetylcholine receptors are also affected by volatile
anesthetics and alcohols, but in opposite directions.
Although acetylcholine receptors can profoundly af-
fect spinal neuronal transmission, block of nicotinic or
muscarinic receptors does not modify anesthetic po-
tency, either in vivo (42,43) or in vitro (44). Thus, in-
hibitory chloride channels, but not acetylcholine re-
ceptors, may be implicated in anesthetic-induced
immobility.
Spinal cord slice preparations allow isolation of
postsynaptic actions on motor neurons from actions on
primary afferents and interneurons. In slices, ethanol
(44) and volatile anesthetics (45) depress currents evoked
by glutamate application, proving that these agents can
directly depress motor neuron excitability. Furthermore,
ethanol (44) and volatile anesthetics (45) depress both
␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) and N-methyl-d-aspartic acid (NMDA) recep-
tor-mediated currents by actions independent of
GABAA and glycine receptors, suggesting that anes-
thetic reduction of motor output can result from depres-
sion of excitation as well as enhancement of inhibition.
Anesthetic effects on the cord are diverse and com-
plex, and the ultimate effect on MAC is incompletely
understood. Spontaneous miniature current frequency
(presynaptically determined), amplitude, and kinetics
(postsynaptically determined) reveal pre- and post-
synaptic anesthetic actions on motor neurons. Halo-
thane, isoflurane, and enflurane prolong glycinergic
current duration but do not increase amplitude (46), in
contrast to nonneuronal expression systems, in which
both amplitude and duration are increased. The three
anesthetics also increase the frequency of spontaneous
transmitter release, but only when sodium channels
are blocked (46). With intact sodium channels, halo-
thane and isoflurane do not affect frequency, and en-
flurane decreases frequency. Changes in charge trans-
fer parallel these effects: increases in presynaptically
mediated frequency plus postsynaptically mediated
prolongation increase the total charge transfer, but
only during sodium channel blockade. That is, in nor-
mal intact preparations, anesthetics do not produce an
absolute increase in inhibition. Thus, for glycinergic
transmission, anesthetics affect the timing rather than
ANESTH ANALG
2003;97:718 –40
the magnitude of inhibition, increasing the period
during which inhibition is effective.
When glutamate AMPA currents are compared
with glycinergic currents, a contrasting picture
emerges. Enflurane decreases miniature current fre-
quency regardless of sodium channel blockade (47)
and depresses current amplitude without changing
kinetics. Thus, volatile anesthetics tend to increase
glycinergic inhibition by two effects: a presynaptic
enhancement of transmitter release and a postsynaptic
prolongation of current duration. A depressant action
on release, probably via sodium channels, counterbal-
ances these inhibitory actions. Enflurane’s actions on
glutamate transmission are purely depressant, both
pre- and postsynaptically. Immobility may result from
a shift toward inhibition in the balance between exci-
tation and inhibition, rather than from an absolute
increase in inhibition.
In summary, results from studies in the isolated
spinal cord suggest that actions involving both excita-
tory (AMPA and NMDA) and inhibitory (glycine and
GABAA) transmission might produce immobility. Ac-
tions on sodium channels and on other channels, in-
cluding those that determine resting membrane poten-
tial, also may be important. Cholinergic receptors play
little or no role.
What Do In Vitro Studies of Isolated
Inhibitory and Excitatory Receptors
Reveal?
Which multiple neuronal signaling systems govern
crucial components of anesthesia, such as immobility?
Drugs that produce anesthesia by acting on a single
signaling system offer powerful clues to the site of
anesthetic action. Propofol, alfaxalone, and etomidate
primarily produce anesthesia by enhancing GABAA
receptor function (i.e., by increasing the effect of a
given concentration of GABA) (48). Ketamine may
produce anesthesia primarily by inhibiting NMDA
receptor function, although it also acts on acetylcho-
line receptors (49). However, blockade of NMDA
alone does not appear to cause immobility (50), so
ketamine probably causes immobility by more than
simply blocking NMDA receptors. Emerging, detailed
molecular analyses of anesthetic actions on GABAA
and glycine receptors and the potential importance of
NMDA receptors prompt the following discussion fo-
cused on these three ion channels (Table 1).
Neurotransmitters signal through two families of
receptors: ionotropic and metabotropic. Ionotropic re-
ceptors are also known as ligand-gated ion channels
because the neurotransmitter (e.g., GABA) binds di-
rectly to the ion channel protein, and this interaction
causes opening (gating) of the ion channel, allowing
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MECHANISMS UNDERLYING MAC
Table 1. Neurotransmitter-Gated Ion Channels as Possible
Targets for Anesthetics
Effect of 1–2 MAC on
expressed receptora
Channel Volatile Gaseous Nonimmobilizer Mutationsb
Inhibitory
GABAA 1 0 0 Yes
Glycine 1 1 0 Yes
Excitatory
NMDA 2/0 2 0/2 No
AMPA 2/0 2 0 No
Kainate 1 2 0 Yes
Nicotinic 22 2 2 Yes
5-HT3 1 ND 0 Yes
MAC ⫽ minimum alveolar anesthetic concentration; GABA ⫽
␥-aminobutyric acid; NMDA ⫽ N-methyl-d-aspartate; AMPA ⫽ ␣-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid; 5-HT ⫽ 5-hydroxytryptamine.
a
Indicates reliable effects of drug concentrations corresponding to one or
two times MAC of volatile inhaled anesthetics (primarily isoflurane, enflu-
rane, sevoflurane, 1-chloro-1,2,2-trifluorocyclobutane [F3], or halothane), gas-
eous inhaled anesthetics (xenon, nitrous oxide, cyclopropane, or butane), or a
nonimmobilizer (primarily 1,2-dichlorohexafluorocyclobutane [F6]). Up ar-
rows indicate enhancement; down arrows indicate block. 0 ⫽ no effect; ND ⫽
no data.
b
Indicates whether mutation of amino acids in the receptor have been
studied and shown to alter the action of volatile anesthetics on the receptor (in
vitro studies) (e.g., Mihic J, personal communication, and Ref. 51).
transmission of specific ions (e.g., chloride ions), with
resulting changes in membrane potential. Ionotropic
receptors are composed of several subunits (e.g., a
pentameric receptor), and each subunit consists of
four transmembrane segments. In contrast, metabo-
tropic receptors are monomeric receptors consisting of
seven transmembrane segments. Binding of neuro-
transmitter (e.g., acetylcholine) to metabotropic recep-
tors causes activation of guanosine triphosphate-
binding proteins (G-proteins) associated with the
receptor, and these G-proteins act as second messen-
gers to activate other signaling molecules, such as
protein kinases, or potassium or calcium channels.
Note that the properties of a receptor may be deter-
mined by the particular subunits from which it is
composed (i.e., not all receptors of a given class [e.g.,
GABAA receptors] are the same).
Ionotropic GABA receptors include GABAA and
GABAC classes. (A metabotropic GABAB receptor cou-
pled through guanosine triphosphate-binding proteins
is not a ligand-gated ion channel.) GABAA receptors
mediate much of the inhibitory neurotransmission in the
central nervous system (CNS) (52). GABAA and GABAC
receptors are formed from combinations of ␣, ␤, ␥, and ␦
subunits, each of which may have multiple types (␣1– 6,
␤1–3, and ␥1–3). Although ␲ and ⑀ subunits also exist,
their function remains obscure. GABAC receptors likely
form from ␳1–3 subunits. Homomeric (all subunits the
same) ␳1 receptors are functional (53,54). Most CNS
GABAA receptors have ␣, ␤, and ␥ subunits. GABA
binding to extracellular sites on the receptor opens a
chloride channel through the receptor. A response to
722 REVIEW ARTICLE SONNER ET AL.
MECHANISMS UNDERLYING MAC
GABA requires both ␣ and ␤ subunits; benzodiazepine
enhancement of GABA function requires ␣, ␤, and ␥
subunits (55).
Two types of subunits (␣ and ␤) form glycine recep-
tors. Only one ␤ subunit has been identified, and ␣1 is
the major ␣ subunit in adults. Homomeric ␣1 or ␣2
receptors are functional (56 –58). As discussed below,
homomeric (e.g., ␳1 or glycine ␣1) receptors expressed
in Xenopus oocytes have advanced studies of molecu-
lar sites of anesthetic action.
Several observations suggest the potential impor-
tance of these receptors to the immobility produced by
inhaled anesthetics. Neuronal and several nonneuro-
nal expression systems reveal that relevant (1 MAC)
concentrations of volatile anesthetics and n-alcohols
potentiate GABAA and glycine receptor function
(52,59 – 63). Most IV anesthetics also potentiate
GABAA receptor function, and some potentiate gly-
cine receptor function (61– 63). Nonimmobilizing con-
geners of several fluorinated anesthetics do not affect
these receptors (52,59 – 61,64).
Comparison of the effects of anesthetics versus non-
immobilizers can be used as one test of the relevance of
a particular ion channel. Although the anesthetic
1-chloro-1,2,2-trifluorocyclobutane (called 1A or F3) and
the structurally similar nonimmobilizer 2N, or F6, have
lipid solubilities consistent with an anesthetic capacity,
only F3 affects the function of GABAA, glycine, AMPA,
kainate, and 5-hydroxytryptamine (5-HT)3 receptors
(59), and thus these receptors pass the test. The nonim-
mobilizer 2,3-dichlorooctafluorobutane (F8) also does
not affect the function of several ligand-gated ion chan-
nels. In contrast, both F3 and F6 inhibit neuronal nico-
tinic receptors and several metabotropic receptors (5-
HT2C, muscarinic-1, mGluR5) (65,66), and thus these
receptors do not pass the test.
How do inhaled anesthetics change the function of
inhibitory neurotransmitter receptors? The differential
actions of anesthetics on two homologous ligand-
gated ion channels allow an exploration of the molec-
ular sites of anesthetic action. In vitro, homomeric
glycine ␣1 and GABA ␳1 receptors respond oppositely
to volatile anesthetics, which enhance function in the
former (61) and inhibit function in the latter (67). A
genetic approach (one that combines various parts of
homomeric glycine ␣1 and GABA ␳1 receptors to pro-
duce new receptors) allows identification of amino
acids in transmembrane segment 2 and in transmem-
brane segment 3 of glycine and GABAA receptors that
permit ethanol enhancement of receptor function (68).
These amino acids lie near the extracellular surface of
the membrane. Results from independent studies sug-
gest the importance of a site near the extracellular
surface (69 –72).
The possibility that propanethiol, an anesthetic in
animals (73), could irreversibly react with cysteines
engineered at those critical positions provided more
ANESTH ANALG
2003;97:718 –40
direct evidence for binding of anesthetics to these
amino acids in glycine and GABAA receptors (74).
Propanethiol and the related sulfhydryl-specific
reagent, propyl methanethiosulfonate (PMTS),
covalently bind to the mutated cysteines and perma-
nently enhance receptor function (i.e., they produce
irreversible anesthetic-like effects.) Competition ex-
periments performed before and after irreversible
binding of PMTS to the cysteine mutations in the
GABAA receptor suggested competition for a single
binding site among PMTS and the inhaled anesthetics
or octanol, but not between PMTS and alfaxalone. This
is consistent with the binding of inhaled anesthetics
and n-alcohols to the same site but the binding of
alfaxalone to a different site (75). Similar conclusions
apply to the glycine receptor.
As noted above, the NMDA receptor also holds
promise as a site of anesthetic action. NMDA receptors
constitute one glutamate receptor subtype, and they
require the coagonists glycine and glutamate for acti-
vation (76). The subunits NR1, NR2 (A-D), and NR3
(A-B) form NMDA receptors (77–79). The NR1 subunit
confers essential function to the NMDA receptor and
is expressed throughout the CNS. In adult mouse
brain, NR1, NR2A, and NR2B subunits are widely
expressed, whereas NR2C occurs predominantly in
the cerebellum, and small levels of NR2D exist only in
the thalamus, brainstem, and spinal cord (80). Most
NR3B subunits are on motor neurons (79), whereas
NR3A has wide distribution (81,82).
The Xenopus oocyte expression system allows study
of receptors with pharmacologic properties that may
mimic those of native neuronal receptors (59). NMDA
receptors expressed from cloned receptors also pro-
vide evidence that inhaled anesthetics depress NMDA
receptors. Clinically relevant concentrations of isoflu-
rane, sevoflurane, and desflurane inhibit recombinant
NR1/NR2A and NR1/NR2B NMDA receptors in a
reversible, dose-dependent, and voltage-insensitive
manner (83). Similarly, enflurane, urethane, nitrous
oxide, xenon, cyclopropane, and butane inhibit
NMDA-stimulated currents in oocytes expressing
NMDA receptors (84 – 87). Thus, considerable evi-
dence shows that a wide range of volatile anesthetics
inhibit NMDA receptor function.
These results indicate that inhaled anesthetic actions
on ligand-gated ion channels may explain the capacity
of inhaled anesthetics to produce immobility; they
point to candidate proteins that might mediate immo-
bility. Whether the associated receptors contribute to
anesthetic-induced immobility can be determined
only from studies in intact organisms. A definitive test
of the importance of anesthetic influence on these
channels could, in principle, be achieved by designing
mutant receptors that provide normal synaptic trans-
mission but resist any anesthetic effect and expressing
ANESTH ANALG
2003;97:718 –40
these receptors in animals. The molecular understand-
ing needed to produce these mutants is emerging for
the GABAA and glycine receptors but is lacking for
most other ligand-gated ion channels. As discussed
elsewhere in this review, the introduction of drug-
resistant receptors into animals (knockin mice) has
provided remarkable insights regarding the actions of
benzodiazepines (88,89) and etomidate and propofol
(90) and will likely prove important for understanding
inhaled anesthetic action.
Genetic Engineering: Definitions and
Implications for Studies of Anesthetic
Mechanisms
Present technologies allow the creation of animals
with genetic modifications that allow tests of specific
receptor targets of anesthetics: transgenic, knockout
(global or conditional), and knockin animals (Table 2).
Transgenic animals overexpress or misexpress an
added gene (referred to as a transgene). They are cre-
ated by microinjection of DNA into the pronucleus of
a recently fertilized embryo. Several disadvantages
limit the usefulness of this older, fast, widely available
technique. Because the added gene inserts randomly
into the genome, expression can be variable, and,
more importantly, the transgene must be dominant
because the animals also have the endogenous gene.
Mating transgenic animals to animals lacking the gene
of interest (not always available) can circumvent this
problem, but this is time consuming.
Creation of animals with precise genetic alterations
requires more sophisticated techniques such as gene
targeting and embryonic stem cell technologies (91).
With this approach, virtually any new endogenous
gene can be created. Mutations rendering specific en-
dogenous genes nonfunctional are “gene knockouts”
(typically for the animal’s lifetime in all cells, a “global
knockout”). Although expensive and time consuming,
this now-routine technology has produced more than
1000 global gene knockouts, many available from mu-
tant mouse repositories (see http://www.jax.org/
imr/index.html). Although global knockouts have ad-
vanced an understanding of anesthetic mechanisms
(90), two problems can confound results from global
knockout studies (41). First, gene inactivation may
induce compensation by altering the expression of
other genes, and this result functionally compensates
for the loss of the targeted gene. Second, it is nearly
impossible to assign a phenotype such as MAC to a
particular neural site because the knockout affects all
neurons.
Conditional knockouts in which specific endoge-
nous genes are inactivated in specific cells or tissues
and/or at specific animal ages circumvent some of
REVIEW ARTICLE SONNER ET AL. 723
MECHANISMS UNDERLYING MAC
these limitations. This complex approach requires
crossing two different lines of genetically engineered
mice. Making mutations in specific cells or neuronal
pathways after key developmental events have oc-
curred minimizes compensation and simplifies the as-
signment of phenotype to neuroanatomic substrates.
Although this approach has successfully dissected as-
pects of learning and memory (92), it has not been
applied to studies of anesthetic mechanisms.
The gene knockin animal differs from a knockout
animal (in which a gene of interest is inactivated) in
that a knockin animal expresses a mutation in the gene
of interest. For example, the gene might now produce
a protein changed by a single amino acid. Because the
normal endogenous promoter controls expression of
the mutant gene, a knockin animal expresses the mu-
tant gene in the same amount, at the same time, and in
the same tissues as expressed by the normal gene. This
approach allowed dissection of the contribution of
individual GABAA-receptor subunits to behavioral re-
sponses induced by benzodiazepines (93). Most im-
portantly, it allowed the construction of mice resistant
to anesthesia from propofol and etomidate (1). It also
may allow the dissection of inhaled anesthetic mech-
anisms. Single amino acid substitutions in individual
GABAA-receptor subunit genes can change the re-
sponse of the receptors in expression systems to alco-
hol and volatile anesthetics without changing the re-
sponse to GABA (68), and mice harboring these
mutations may allow for unambiguous testing of the
contribution of specific receptor subunits to MAC.
Such genetically engineered mice are currently being
tested (94). However, the inhaled anesthetics may
present a challenge not offered by anesthetics such as
propofol or etomidate. Probably only one receptor
(GABAA) mediates the effects of propofol and etomi-
date, but multiple receptors may mediate the effects of
inhaled anesthetics. Thus, results from a knockin in-
volving a single receptor may elucidate only one as-
pect of inhaled anesthetic actions.
What Do In Vivo Studies Reveal
Concerning the Ion Channels Mediating
MAC?
Pharmacologic approaches can complement the previ-
ously described genetic approaches to reveal the rele-
vance of candidate receptors to MAC. Both assume
that altering the function of a relevant receptor will
change MAC. The genetic approach determines the
effect of receptor mutations (e.g., elimination or mod-
ification of a specific receptor) on MAC. The pharma-
cologic approach determines the effect of agonists or
antagonists of specific ion channels on MAC.
Genetic manipulations and pharmacologic treat-
ments can change MAC directly or indirectly. For
724 REVIEW ARTICLE SONNER ET AL. ANESTH ANALG
MECHANISMS UNDERLYING MAC 2003;97:718 –40

Table 2. Genetically Engineered Animals

Animal type Purpose Advantages Disadvantages
Transgenic Overexpress or Relatively fast; least technically Random insertion, variable
misexpress gene demanding expression, transgene must
have dominant effect or be bred
onto knockout background
Global gene Inactivate gene in all cells Now routine; ⬎1000 strains Slow and expensive, technically
knockout of body at all times available demanding, developmental
compensation, nearly
impossible to attribute specific
phenotype to specific cell type,
circuit, or brain region
Conditional Inactivate gene in specific Can limit knockout to specific Slow and expensive, technically
knockout cells and tissues and/ tissue and/or time, minimizes demanding, not presently
or at specific times compensation, many strains possible to target every tissue
currently under development, or time point of interest
can mix and match mouse
strains to achieve desired
knockout
Gene knockin Normally express mutant Compensation absent or Slow and expensive, technically
gene from endogenous minimized demanding
control elements

example, consider the action of two drugs on heart
rate. Epinephrine acts directly on the sympathetic
nerve terminals to increase heart rate. Atropine also
increases heart rate but does so indirectly by blocking
the action of acetylcholine on the parasympathetic
nervous system. Similar considerations apply to MAC.
Although opioids decrease MAC, inhaled anesthetics
do not act via opioid receptors (see below). Opioid
receptors can play an indirect (modulating) role but
not a direct (mediating) role. This critical distinction
will become apparent in the discussion of the role of
the GABAA receptor in MAC. A direct effect reflects a
mediating role for an ion channel (the terms “direct
effect” and “mediating role” are interchangeable; see
Fig. 3).
As described previously, the most powerful genetic
approach to this issue develops mice with subtle site-
directed mutations that retain the normal function of
the receptor but abolish the capacity of anesthetics to
affect normal function. For a mechanistically relevant
receptor, such an alteration should produce an appar-
ently normal animal that, compared with mice with-
out the mutation, moves in response to noxious stim-
ulation at inhaled anesthetic concentrations markedly
exceeding those that normally produce immobility.
Pharmacologic approaches can also be used to dis-
tinguish between direct and indirect effects of receptor
systems on MAC. If the administration of a receptor
antagonist produces no measurable effect on MAC,
then that receptor neither directly nor indirectly af-
fects MAC and is not relevant. But suppose an antag-
onist has an effect? Because the spinal cord mediates
MAC, an intrathecal (spinal) dose of antagonist should
change MAC more than the same or larger dose given
IV. If it does not, then the antagonist effect is probably
supraspinal and not directly relevant to MAC.
However, perhaps an antagonist affects MAC, and
the systemic dose required for a given change exceeds
the intrathecal dose. To be relevant, the receptor must
pass one more test. To be a relevant receptor (one
directly acted on to produce the anesthetic effect), the
injection of an antagonist should change MAC in pro-
portion to the in vitro capacity of each anesthetic to
affect the receptor. The power of this approach is
suggested in the “proof-of-principle” experiment
shown in Figure 4. In rats, the noncompetitive GABAA
antagonist picrotoxin increases the immobilizing ED50
of the non-GABAergic anesthetic ketamine to a ceiling
of approximately 60% (reflecting an indirect antago-
nism of the effect of naturally occurring tonic GABA
release) but increases the ED50 of the GABAergic an-
esthetic propofol by approximately 400% and shows
no ceiling effect (primarily reflecting a direct antago-
nism) (49,95). In this study, isoflurane acts like ket-
amine, the non-GABAergic anesthetic. Gabazine (a
competitive antagonist at GABAA receptors) has the
same effect as picrotoxin.
The genetic and pharmacologic approaches are
complementary. When different mutations and
drug treatments show the same pattern (e.g., of no
discernible effect), the consistency of findings
strengthens the conclusions regarding the relevance
of the target receptor. One caveat applies to both
genetic and pharmacologic tests of the relevance of
a specific ion channel: if two systems must be
blocked concurrently to produce immobility, then
blocking or knocking out just one may not provide
an adequate test.
ANESTH ANALG
2003;97:718 –40
Figure 3. This drawing illustrates the difference between the site (or
sites) that directly mediates the actions of inhaled anesthetics versus
sites that may influence inhaled anesthetic potency indirectly by
modulating the function of the true (directly affected) site (or sites)
of action. Modulation may occur through an action on the mediat-
ing site or by affecting the input to or output from that site. For
example, a tonic release of ␥-aminobutyric acid (GABA) from an
indirect site might affect the measured minimum alveolar anesthetic
concentration (MAC) of all inhaled anesthetics by inhibiting activity
in the anesthetic site of action. Blocking this input would increase
MAC, but this increase would not mean that GABAA receptors
mediate inhaled anesthetic actions.
Inhibitory Ionotropic Ligand-Gated and
Voltage-Gated Channels
Glycine. Glycine receptors are major mediators of
inhibitory neurotransmission in the spinal cord. Their
spinal localization and their potentiation by clinically
important volatile anesthetics make them prime can-
didates as mediators of MAC. Evidence in animals
supports this conjecture. IV and intrathecal adminis-
tration of strychnine, a glycine receptor antagonist,
increases MAC (96,97). The MAC increase for cyclo-
propane, isoflurane, and halothane produced by intra-
thecal strychnine administration correlates with the in
vitro enhancing effect of these anesthetics on the gly-
cine receptors (97) (Fig. 5) (98), a result expected if the
enhancing action on the glycine receptor mediates
part of the immobilizing effect of these anesthetics.
Mice with mutations in the glycine receptor show a
complex and inconsistent pattern of glycine effects.
Spastic mice have decreased expression of the glycine
receptor because of abnormal RNA splicing of the ␤
subunit (99). Compared with controls, spastic mice
have a 30% increase in enflurane MAC but no increase
REVIEW ARTICLE SONNER ET AL. 725
MECHANISMS UNDERLYING MAC
Figure 4. In rats, IV infusion of the noncompetitive ␥-aminobutyric
acid (GABA)A antagonist picrotoxin increases the immobilizing 50%
effective dose (ED50) of the non-GABAergic anesthetic ketamine to
a ceiling of approximately 60%. Because ketamine is not known to
materially influence GABAA receptors, this effect of picrotoxin
probably reflects an indirect antagonism of the effect of naturally
occurring tonic GABA release. Picrotoxin similarly increases the
immobilizing ED50 (minimum alveolar anesthetic concentration;
MAC) of isoflurane. In contrast, picrotoxin increases the ED50 of the
GABAergic anesthetic propofol by approximately 400% and shows
no ceiling effect (primarily reflecting a direct antagonism) (49,95).
The reader will note that 0.5 MAC isoflurane was added in both the
ketamine and the propofol studies. This was done because the
administration of picrotoxin in the absence of isoflurane was lethal
in animals anesthetized with ketamine. The administration of isoflu-
rane prevented death. Similar results were obtained when the
isoflurane concentration was 0.25 MAC.
in halothane MAC (100). Spasmodic mice have de-
creased sensitivity to glycine because of a missense
mutation in the glycine receptor ␣ subunit (101), but
they show no change in MAC to enflurane or halo-
thane (100). Interpretation of these results with mutant
mice may be compromised by the development of
compensatory changes (see above).
GABAA. Although they mediate the immobility pro-
duced by injectable anesthetics (propofol and etomi-
date), GABAA receptors may not mediate the immobility
produced by inhaled anesthetics. The proof-of-principle
experiment illustrated in Figure 4 indicates that isoflu-
rane resembles the non-GABAergic anesthetic ketamine,
but not the GABAergic anesthetic propofol. This sug-
gests that isoflurane’s immobilizing effect is not directly
mediated by GABAA receptors.
Similar conclusions follow from results of studies
applying intrathecal picrotoxin to rats inhaling isoflu-
rane versus xenon or cyclopropane, anesthetics with
minimal effects on GABAA receptors (Zhang Y, un-
published data) (Fig. 5). If inhaled anesthetic enhance-
ment of the response of GABAA receptors directly
mediates the capacity of inhaled anesthetics to sup-
press movement in response to noxious stimuli, then
blockade of GABAA receptors should increase the
MAC of isoflurane more than the MAC of xenon or
cyclopropane. However, MAC increases equally for
all three anesthetics, indicating that GABAA receptors
are no more important to the immobilizing action of
isoflurane than for anesthetics with minimal effects on
726 REVIEW ARTICLE SONNER ET AL.
MECHANISMS UNDERLYING MAC
Figure 5. Administration of strychnine, a glycine receptor antago-
nist, increases minimum alveolar anesthetic concentration (MAC)
for rats (96). The MAC increase for cyclopropane, isoflurane, and
halothane produced by intrathecal strychnine administration corre-
lates with the in vitro capacity of these anesthetics (at a concentra-
tion equal to MAC) to enhance (increase) the response of glycine
receptors to glycine (97). Each point is the measured increase in
MAC of a rat at the corresponding enhanced receptor activity
calculated from pooled in vitro results (85,97). In contrast, although
administration of picrotoxin, a ␥-aminobutyric acid (GABA)A an-
tagonist, also increases MAC for cyclopropane, xenon, and isoflu-
rane, the increase in MAC does not correlate with the in vitro
enhancing effect of these anesthetics (98) (data for xenon and halo-
thane were not available, respectively, for glycine and GABAA
receptors).
GABAA receptors. It may be argued that the in vitro
data for the effective concentration of anesthetics may
be influenced by many variables (e.g., agonist concen-
tration, use of peak current, or current decay) and that
the studies of Zhang selected inappropriate in vitro
concentrations. However, that argument ignores the
finding that the dose-response relationship in Figure 5
is flat (i.e., it almost does not matter where the in vitro
data lie on the abscissa).
Other evidence supports the conclusion that the
enhancing effect of volatile anesthetics on GABAA
receptors minimally influences MAC. Knockout of the
␤3 subunit of GABAA increases enflurane MAC by
26% but increases halothane MAC by only 9% (102),
and even these small increases may be attributable to
compensation for lack of the subunit (41). Such results
for inhaled anesthetics quantitatively agree with those
found by Jurd et al. (1) for ␤3 N265M knockin mice
unresponsive to the anesthetic effects of propofol: en-
flurane MAC increased by 15% and halothane MAC
by 21%. The mutation knocked into the mice had
normal in vitro sensitivity to enflurane and halothane.
ANESTH ANALG
2003;97:718 –40
Figure 6. In rats, blockade of the 5-hydroxytryptamine-3 receptor by
systemic (114) (Fig. 6) or intrathecal (Flood, unpublished data)
administration of ondansetron does not affect the minimum alveo-
lar anesthetic concentration (MAC) of isoflurane, even at doses of
ondansetron modestly below the lethal dose. These findings indi-
cate that this receptor does not directly mediate the immobility
produced by inhaled anesthetics.
The MAC for a series of fluorinated alkanols (103)
bears no consistent relationship to their capacity to
enhance the effect of GABA on GABAA receptors
(104). Enflurane and halothane enhance GABA-
mediated chloride conductance in rat hippocampal
neurons (105), but a given MAC multiple of enflurane
has twice the effect of halothane (i.e., the result is not
quantitatively consistent across anesthetics.)
In summary, anesthetizing concentrations of many
inhaled anesthetics enhance the in vitro action of
GABA as much as do etomidate and propofol, anes-
thetics thought to produce immobility solely by en-
hancing the action of GABA on GABAA receptors.
Also, studies of ␤3 knockout and knockin mice indi-
cate that GABAA receptors may have a mediating role,
albeit a minor one, in immobility. These findings sug-
gest that enhancement of GABA actions should un-
derlie at least part of the immobility produced by
inhaled anesthetics. However, drugs that block
GABAA receptor function in vivo do not markedly
increase MAC for inhaled anesthetics; the increase
parallels that for ketamine, an anesthetic not thought
to act by enhancement of GABAA receptors; the in-
crease in MAC is the same for inhaled anesthetics that
do and do not enhance the effect of GABA in vitro;
and, similarly, the capacity of fluorinated alcohols to
enhance the effect of GABA does not predict their
potency. Thus, in vivo results suggest a minor or no
role for GABAA receptors as mediators of immobility,
whereas in vitro receptor studies and parallels with
anesthetics such as propofol support a role. Although
GABAA receptors are unlikely mediator candidates, a
conclusive determination may require the develop-
ment of knockin mice with GABAA receptors that
ANESTH ANALG REVIEW ARTICLE SONNER ET AL. 727
2003;97:718 –40 MECHANISMS UNDERLYING MAC

respond normally to GABA but are not potentiated by

inhaled anesthetics.
Potassium Channels. Because potassium channels
are numerous and diverse and because an increase in
potassium conductance can decrease the excitability of
the nervous system, potassium channels are plausible
candidates as mediators of the immobility produced
by inhaled anesthetics. Franks and Lieb (106) found
that inhaled anesthetics potentiate potassium leak
channels, and therefore reduce neuronal excitability,
in the pond snail genus Lymnaea. Mammalian potas-
sium channel subunits of the KCNK (K is potassium,
CN is channel, and K is the subfamily of potassium
channels) subfamily of leak channels (channels whose
currents are not strongly gated by voltage) have prop-
erties similar to the properties of the Lymnaea channel, Figure 7. Administration of nicotinic (mecamylamine) or muscarinic
including activation by inhaled anesthetics (107–110). (atropine or scopolamine) antagonists to rats does not decrease the
Although the KCNK subfamily provides a plausible isoflurane concentrations required to produce immobility (42,43).
These and other data indicate that cholinergic inhibition by volatile
anesthetic target, are there any holes in this leak compounds does not mediate anesthetic-induced immobility. MAC
theory? ⫽ minimum alveolar anesthetic concentration.
No present evidence demonstrates that potassium
channels directly mediate immobility. Only one (neg-
one a ligand-gated ion channel (nicotinic or nicotinic
ative) study of inhaled anesthetic potency in potas-
acetylcholine receptor [nAChR]) and the other a
sium channel knockout mice has been reported (111).
G-protein-linked (muscarinic) receptor. Nicotinic re-
One study of the nonspecific KCNK activator riluzole
ceptors expressed in neurons have been suggested as
supports the hypothesis that, although this drug has
putative mediators of general anesthetic actions be-
anesthetic properties, this action occurs in the brain
cause small concentrations of volatile anesthetics and
and not the spinal cord (Gray A, Zhang Y, unpub-
ketamine inhibit many such receptors and because the
lished data). That is, IV and intrathecal infusions of
(⫹) isomer of isoflurane is approximately 50% more
riluzole that do not produce permanent injury or
potent than the (⫺) isomer at inhibiting MAC in rats
death do not decrease isoflurane MAC, indicating that
(115), and the same ratio of potency has been observed
KCNK channels do not mediate MAC. In summary,
at molluscan nAChRs (116).
present evidence does not indicate that a specific po-
If antagonism of nicotinic receptors by volatile an-
tassium channel (or channels) mediates the immobil-
esthetics decreases motor responses to noxious stim-
ity produced by inhaled anesthetics, but the large
ulation, the administration of a nicotinic antagonist
number and diversity of potassium channels pre-
(mecamylamine) to rats should, but does not (Fig. 7),
cludes a conclusion that they play no role.
decrease the volatile anesthetic concentrations re-
quired to produce immobility (42,43). Similarly, the
Excitatory Ionotropic Ligand-Gated and administration of the classic agonist nicotine (at con-
Voltage-Gated Channels centrations known to have behavioral affects attrib-
uted to nicotinic modulation) (117) does not alter the
5-HT3 Receptors. Although most 5-HT receptors
MAC of isoflurane (42). Finally, nonimmobilizers (in-
couple to second-messenger systems, potentially lead-
haled compounds predicted from their hydrophobic-
ing to various downstream effects, the 5-HT3 receptor
ity to have anesthetic activity that do not cause immo-
is a cationic ionophore with considerable sequence
bility alone or add to the anesthesia provided by
homology to GABAA, glycine, and nicotinic acetylcho-
known anesthetics) (5) inhibit nAChRs (118). Such
line receptors. Thus, not surprisingly, some inhaled
observations suggest that nAChRs do not mediate
anesthetics potentiate the ionophoric response of the
inhaled anesthetic-induced immobility.
5-HT3 receptor (112,113). This in vitro proexcitatory
effect of some anesthetics suggests that the 5-HT3 re- Glutamate (NMDA, AMPA, and Kainate)
ceptor cannot mediate immobility. Consistent with
this conclusion, in rats, blockade of the 5-HT3 receptor NMDA Receptors. As noted above, glutamate is
by systemic (114) (Fig. 6) or intrathecal (Flood, unpub- the major mammalian excitatory neurotransmitter in
lished data) administration of ondansetron does not the CNS. Glutamate receptors include metabotropic
affect the MAC of isoflurane. G-protein-coupled receptors and three ligand-gated
Neuronal Nicotinic Acetylcholine Receptors. There ion channel classes: NMDA, AMPA, and kainate re-
are two types of acetylcholine receptors in the CNS: ceptors (119,120).
728 REVIEW ARTICLE SONNER ET AL.
MECHANISMS UNDERLYING MAC
Numerous results suggest the potential importance
of NMDA receptors as mediators of the immobilizing
capacity of inhaled anesthetics. Diethyl ether, chloro-
form, methoxyflurane, halothane, enflurane, and
isoflurane decrease glutamate-stimulated binding of
MK-801 to the NMDA receptor (MK-801 [also called
dizocilpine] is a standard blocker of NMDA recep-
tors). Application of glutamate normally increases
binding of MK-801, which thereby serves as an indi-
cation of NMDA receptor activity. Thus, a decrease of
binding produced by the volatile anesthetics indicates
decreased NMDA receptor activity (121). Inhaled an-
esthetics inhibit NMDA receptors in receptor expres-
sion systems (87). Nitrous oxide and xenon have
greater in vitro effects than potent inhaled anesthetics
on NMDA receptors (122), and their actions on
NMDA receptors and/or sodium channels may con-
tribute more to their immobilizing capacity than is the
case with anesthetics such as isoflurane. As discussed
above, both enflurane (45) and ethanol (44) can de-
crease NMDA-mediated glutamate currents in motor
neurons in the spinal cord slice, an effect independent
of effects on GABAA and glycine receptors.
Masaki et al. (123) applied the NMDA antagonist D
(⫺)-2-amino-5-phosphonopentanoic acid (D-AP5)
intracerebroventricularly and noted a sevoflurane
MAC-sparing effect, suggesting a supraspinal effect.
Ishizaki et al. (124,125) found a maximal decrease of
30% in isoflurane MAC in rats given intrathecal bolus
doses of the NMDA antagonists AP5, MK-801, CPP,
and 7CKA. McFarlane et al. (126) reported an 80%
decrease in MAC of halothane from IV application of
the competitive NMDA receptor antagonist CGS
19755.
MK-801 administration to rats decreases isoflurane
MAC, and the decrease primarily correlates with MK-
801 concentrations in the spinal cord, rather than
whole brain or cerebral cortex concentrations, a find-
ing consistent with mediation of the decreased MAC
by the cord (50). Do inhaled anesthetics produce ef-
fects similar to MK-801: do they block NMDA recep-
tors so as to produce immobility (i.e., do NMDA re-
ceptors in the spinal cord directly mediate MAC)?
Temporal summation is the cumulative effect of a
succession of repeated stimuli presented at sufficiently
close intervals (127). Phrased another way, a shorten-
ing of the intervals between stimuli increases the col-
lective stimulation and is more likely to provoke a
response. In vitro, blocking NMDA receptors blocks
temporal summation, indicating that NMDA recep-
tors lie in the pathway that mediates temporal sum-
mation (128). These observations suggest that persis-
tence of temporal summation during anesthetic
administration reflects intact NMDA receptor func-
tion. Studies with isoflurane suggest that approxi-
mately 40% of the generation of movement evoked by
ANESTH ANALG
2003;97:718 –40
Figure 8. Isoflurane concentrations (mean ⫾ sd) required to produce
immobility in response to noxious stimulation (intermittent 50-V,
0.5-ms square-wave pulses) increase with decreasing interstimulus
interval (ISI) (127). The concentration required to suppress move-
ment at an ISI of 0.1 s does not differ from the minimum alveolar
anesthetic concentration (MAC). MK-801 (an N-methyl-d-aspartate
antagonist that impairs temporal summation) administration added
to isoflurane administration prevents shorter ISIs from provoking
movement at larger isoflurane concentrations.
noxious stimulation (MAC) depends on the inter-
stimulus interval, suggesting the persistence of tem-
poral summation and transmission via NMDA path-
ways (Fig. 8). Strengthening this interpretation, if
temporal summation persists, then the administration
of the NMDA blocker MK-801 should abolish summa-
tion, and that is what appears to occur (Fig. 8). Also
consistent with the interpretation that isoflurane does
not block temporal summation, electrophysiologic
studies of neuronal windup show that temporal sum-
mation can occur during anesthesia (129). However,
another interpretation is possible: perhaps isoflurane
causes suppression of NMDA receptor transmission,
and blockade with MK-801 simply substitutes for the
blockade produced by isoflurane.
In contrast to the findings with isoflurane, temporal
summation is muted during anesthesia with xenon
(Dutton R, unpublished data). The greater effect of
xenon is consistent with its potent in vitro capacity to
block NMDA receptors (87,122).
In summary, evidence suggesting the importance of
NMDA receptors to MAC include the observations
that (1) in vitro, inhaled anesthetics can block NMDA
receptors; (2) blockade of NMDA receptors (e.g., with
MK-801) decreases MAC; and (3) this decrease in
MAC correlates with the concentration of MK-801 in
the lower part of the spinal cord. However, studies
indicating that temporal summation persists during
isoflurane anesthesia and that MK-801 blocks such
summation argue against a role for NMDA receptors
ANESTH ANALG
2003;97:718 –40
as mediators of isoflurane MAC. Conversely, the con-
trasting data for xenon and isoflurane suggest the
possibility that NMDA receptors might mediate a por-
tion of the capacity of some anesthetics (xenon), but
not others (isoflurane), to produce immobility. None
of these results excludes a role for NMDA receptors in
the mediation of other aspects of anesthesia, including
the capacity of inhaled anesthetics to impair learning
and memory (i.e., produce amnesia).
AMPA Receptors. AMPA receptors mediate the fast
(initial) component of excitatory postsynaptic trans-
mission and provide plausible targets for volatile an-
esthetics. Such fast transmission contrasts with the
slower (later) excitatory transmission mediated by
NMDA receptors. Competitive antagonists for AMPA
receptors, administered IV (130) or intrathecally (124),
markedly decrease the concentration of halothane that
causes immobility, findings consistent with the medi-
ation of anesthetic-induced immobility by AMPA re-
ceptors. However, although showing that movement
requires AMPA receptor function, these results do not
prove or disprove that anesthetics impair AMPA re-
ceptor function. Indeed, one study found that intra-
thecal antagonists fail to affect isoflurane MAC (131),
but this study used smaller doses of antagonist than in
the study that did show a decrease.
In vitro electrophysiologic studies demonstrate that
volatile anesthetics inhibit AMPA-mediated excitatory
postsynaptic currents, that enflurane inhibits the
postsynaptic action of glutamate on AMPA receptors
in mouse spinal cord (45), and that halothane similarly
affects the hippocampus at larger MAC values (25).
Also, clinically relevant concentrations of volatile an-
esthetics partially inhibit native and recombinant
AMPA receptors activated by exogenous agonists
(45,87,132–134). Whether minor inhibition of AMPA
receptors materially influences neuronal network ac-
tivity remains unclear.
Genetically modified mice provide insights into the
importance of inhibition of AMPA receptors to volatile
anesthetic actions. GluR1– 4 subunit combinations form
AMPA receptors. Mice lacking the GluR2 subunit have
inconsistent phenotypic responses to inhaled anesthet-
ics. They show a greater sensitivity to halothane, isoflu-
rane, and sevoflurane than wild-type littermates for loss
of the righting reflex and antinociception but no differ-
ence in MAC (133). In electrophysiologic studies, clinical
concentrations of isoflurane and halothane minimally
inhibit AMPA receptors (both GluR2-containing and
-deficient) (133). Therefore, blockade of AMPA receptors
cannot underlie enhanced sensitivity (for righting reflex
and antinociception) in GluR2 knockout mice. Other ev-
idence suggests that decreased excitatory neurotrans-
mission due to altered AMPA receptor kinetics renders
GluR2-deficient neurons more sensitive to anesthetics
acting on other target receptors (135).
REVIEW ARTICLE SONNER ET AL. 729
MECHANISMS UNDERLYING MAC
Thus, studies using GluR2 knockout mice support
the postulate that different neuronal circuits mediate
MAC versus antinociception and loss of the righting
reflex (133). Motor neurons in the ventral spinal cord
(i.e., that might mediate MAC) lack GluR2 subunits
(136 –138), but neurons that mediate the righting reflex
and nociception do express GluR2-containing AMPA
receptors. Thus, knockout of the GluR2-containing re-
ceptors should not change MAC but might change the
righting reflex and antinociception. Furthermore, neu-
ronal circuits in the spinal cord that mediate MAC do
not appear to require the GluR2 subunit. In summary,
although in vivo data suggest that ablation of the
GluR2 subunit does not affect MAC, this cannot ex-
clude the possibility that other subunits of AMPA
receptors contribute to MAC.
Kainate Receptors. Combinations of GluR5–7, KA1,
and KA2 subunits form the kainate subtype of iono-
tropic glutamate receptors (139). In vitro, inhaled an-
esthetics enhance currents mediated by kainate recep-
tors containing GluR6 (51). However, GluR6 knockout
mice have a normal isoflurane MAC (Sonner, unpub-
lished data). These findings are difficult to interpret
because kainate receptors can be assembled from
other kainate subunits, even in the absence of the
GluR6 subunit. Perhaps other forms of GluR6 muta-
tion, such as GluR6 editing mutant mice (140), will
provide insights that a knockout may not. Presently,
the importance of kainate receptors to MAC remains
speculative.
Sodium Channels. A lingering dogma asserts that
clinical concentrations of inhaled anesthetics do not
block axonal conduction and, thus, voltage-dependent
sodium channels. However, more recent data show
that anesthetics can inhibit presynaptic terminal re-
lease of neurotransmitters, particularly glutamate
(141). Inhibition of these presynaptic sodium channels
could produce this action. A tantalizing finding is that
systemic injection of lidocaine decreases MAC in ani-
mals (142,143) and humans (144). The effect has a floor
(a maximum decrease of 40%–50%) (143) and thus
cannot simply result from blockade of all nerve
conduction.
Molecular cloning provides a basis for reconciling
old ideas about axon conduction with anesthetic inhi-
bition of sodium channel function. Cloning reveals
nine different sodium channel ␣ subunits and three ␤
subunits differentially localized in the brain and the
periphery. Different members of this family may vary
in their anesthetic sensitivity. Several volatile anes-
thetics, including 1A, or F3, may inhibit synaptosomal
sodium channels (141,145–156), but the nonimmobi-
lizer 2N, or F6, does not inhibit (157). Thus, sodium
channels pass this test of anesthetic relevance. Studies
of volatile anesthetic action on cloned sodium chan-
nels show somewhat divergent results that, in part,
730 REVIEW ARTICLE SONNER ET AL.
MECHANISMS UNDERLYING MAC
may be due to differences in the origin (neural versus
nonneural origin of the channel) (148,158,159).
Thus, consistent with the notion that sodium chan-
nels directly mediate anesthesia, several inhaled anes-
thetics, but not the nonimmobilizer F6, inhibit sodium
channels. Key questions remain: which sodium chan-
nel subunits exhibit sensitivity or resistance to anes-
thetics, and what is the neuroanatomical localization
of the sensitive channels? Finally, what is the mecha-
nism of channel inhibition— do anesthetics act directly
on the protein or act through protein kinase C (PKC)
or other modulators?
Metabotropic Receptors
Much of the preceding discussion focused on ion
channels because so much is known about inhaled
anesthetic effects on these receptors. In contrast to ion
channels, metabotropic receptors act through second
messengers released by an intracellular component
such as the G-protein coupled to the receptor (160).
Often, these effects are prolonged, lasting seconds to
minutes. They influence the electrophysiologic prop-
erties of a neuron, including membrane resting poten-
tials (160). As noted below, changes in some of these
receptors (e.g., 5-HT2, opioid, and ␣2 adrenoreceptors)
can significantly decrease MAC. However, of these
examples, only the 5-HT2 receptor might directly me-
diate the capacity of inhaled anesthetics to produce
immobility.
Neuronal Muscarinic Acetylcholine Receptors. Isoflu-
rane inhibits muscarinic receptors through the activa-
tion of PKC (117). If muscarinic antagonism by volatile
anesthetics decreases motor responses to noxious
stimulation, muscarinic antagonists (e.g., atropine or
scopolamine) given to rats should, but do not (Fig. 7),
decrease the volatile anesthetic concentrations re-
quired to produce immobility (43), and intrathecal
administration of atropine does not alter the MAC of
isoflurane (43). Even the combination of muscarinic
and nicotinic blocking drugs does not change MAC
(Fig. 7). Thus, the hypothesis that cholinergic inhibi-
tion by volatile compounds mediates anesthetic-
induced immobility cannot be correct.
Opioid Receptors. Analgesia constitutes a nominal,
albeit sometimes contested, feature of the anesthesia
provided by inhaled anesthetics. Thus, it would be
reasonable to suppose that inhaled anesthetics en-
hance the release of endogenous opioids and that this
release contributes to the anesthetic state. However,
inhaled anesthetics do not appear to increase endog-
enous opioids in cerebrospinal fluid (161), and they do
not suppress autonomic or ventilatory responses to
surgical stimulation at concentrations that suppress
movement (162). Also, small doses of opioids mark-
edly decrease inhaled anesthetic concentrations that
prevent movement (163). That is, the opioids supply
ANESTH ANALG
2003;97:718 –40
Figure 9. In rats, the administration of enormous doses of naloxone,
a drug that blocks the effect of opioids, does not increase (or
decrease) the minimum alveolar anesthetic concentration (MAC) of
halothane (164). If opioid receptors mediated the effect of inhaled
anesthetics, administration of naloxone would be expected to in-
crease MAC. Parallel findings of lack of an effect of naloxone have
been found for nitrous oxide (165).
something (analgesia) that inhaled anesthetics do not.
Finally, the administration of enormous doses of nal-
oxone, a drug that blocks the effect of opioids, has no
effect on the MAC of halothane (Fig. 9) (164) or nitrous
oxide (165) in rodents.
␣2 Adrenoreceptors. Several findings suggest the pos-
sibility that ␣2 adrenoreceptors might mediate the im-
mobility produced by inhaled anesthetics. The adminis-
tration of ␣2-adrenoreceptor agonists decreases the MAC
of inhaled anesthetics in rats (166,167) and dogs
(168,169). The ␣2-adrenoreceptor agonist clonidine de-
creases anesthetic requirements in humans (170–172).
␣2-Adrenoreceptor agonists suppress nociceptive neuro-
transmission in the neonatal rat spinal cord, probably by
depressing substance P and glutamate-mediated path-
ways (39,173). Spinal ␣2 adrenoreceptors may mediate a
portion of the antinociceptive effect of medullary and
spinal ␦2-opioid receptors (174). Intrathecal or epidural
injection of dexmedetomidine in the dog has a far more
powerful antinociceptive effect than that achieved with
intracisternal or IV injection (175). In rats, intrathecal
injection of the ␣2-adrenoreceptor agonist dexmedetomi-
dine produces a dose-dependent inhibition of nocicep-
tive C and innocuous A ␤ responses of dorsal horn
neurons to transcutaneous electrical stimulation (176).
However, depletion of ␣ 2 adrenoreceptors by
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
does not change halothane MAC in rats (167). Also,
ANESTH ANALG
2003;97:718 –40
Figure 10. Administration of yohimbine 0.8 –1.0 mg/kg and atipa-
mezole (␣-adrenoreceptor-blocking drugs) increases the minimum
alveolar anesthetic concentration (MAC) of isoflurane in rats by
approximately 10%. However, still greater doses do not increase
MAC (177). The small (10%) increase in MAC probably results from
suppression of the effect of normal tonic stimulation of ␣2 adreno-
receptors. The absence of a material increase in MAC argues against
a role of ␣-adrenoreceptors as mediators of MAC.
the ␣-adrenoreceptor-blocking drug tolazoline at a
single dose of 5 mg/kg does not affect halothane MAC
in dogs (168). Yohimbine 0.8 –1.0 mg/kg and atipam-
ezole (␣-adrenoreceptor-blocking drugs) increase the
MAC of isoflurane in rats by approximately 10%, but
still larger doses do not increase MAC (177) (Fig. 10).
The small (10%) increase in MAC probably results
from suppression of the effect of normal tonic stimu-
lation of ␣2 adrenoreceptors. Thus, ␣-adrenoreceptors
do not mediate the capacity of inhaled anesthetics to
produce immobility in the face of noxious stimulation.
5-HT2A Receptors. Inhaled anesthetics can block the
in vitro effect of 5-HT on 5-HT2A receptors (65), doing
so at concentrations of approximately 1 MAC. 5-HT2A
receptors may participate in nociceptive processes, as
shown in several studies using a specific blocker of
5-HT2A receptors: ketanserin (178 –186). Blockade with
ketanserin decreases evidence of nociception. Al-
though many of these studies focused on the supraspi-
nal actions of 5-HT and ketanserin, some results also
suggested a direct spinal effect (182,185,187).
Although Dringenberg (188) found no anesthetic
effect after intraperitoneal administration of ketan-
serin 700 ␮g/kg in sheep, Doherty et al. (189) found
that the antagonist R51703 decreased halothane MAC
in dogs. Similarly, Zhang et al. (190) found maximum
decreases in isoflurane MAC in rats of approximately
60%. Larger doses were lethal. Intrathecal administra-
tion of ketanserin produced smaller (20%–25%), but
significant, maximum decreases in MAC. These data
REVIEW ARTICLE SONNER ET AL. 731
MECHANISMS UNDERLYING MAC
are consistent with the idea, but do not prove, that
5-HT2A receptors may directly mediate a small portion
of the capacity of inhaled anesthetics to produce im-
mobility. However, in vitro studies find that the 5-HT2
receptor is equally affected by the nonimmobilizer F6
and by halothane at concentrations predicted to equal
1 MAC (65).
Other Metabotropic Receptors. Little is known con-
cerning the capacity of other metabotropic receptors
(e.g., GABAB and neurokinin receptors) (191) to me-
diate immobility. GABAB receptor activation can hy-
perpolarize dorsal horn neurons, produce antinoci-
ception, and decrease motor neuron activity (192–195).
Although such effects suggest that enhanced GABAB
activity might decrease MAC, the only reported effect
is that a GABAB antagonist did not significantly alter
a measure related to MAC (196). However, the dose of
antagonist was carefully chosen to avoid touch-
evoked allodynia in the awake rat and thus may have
been too small to provide an adequate test. The
neurokinin-1 receptor and its chief ligand, substance
P, mediate nociception. Genetic deletion of this recep-
tor impairs windup of dorsal horn neurons (197).
Windup may be defined as an increase in the number
of action potentials elicited by each stimulus as a train
of stimuli progresses. Impairment of windup may re-
sult from suppression of processes involved in tempo-
ral summation, an effect that could also decrease MAC
(127,198,199). Metabotropic glutamate receptors are
less likely to be involved in immobility because the
Class I receptors (types 1 and 5), the forms most
frequently found in the spinal cord, are equally af-
fected by the nonimmobilizer F6, whereas anesthetics
only indirectly affect type 5 and do not affect type 1
(66,191).
The Place of Molecular Modeling in
Future Studies
How do anesthetics interact with ion channels? Mo-
lecular modeling contributes to theories of anesthesia
at three atomic levels: modeling of site-directed mu-
tations, molecular dynamics simulations of anesthetics
in membranes, and modeling of the structure of
ligand-gated ion channels.
Single amino acid mutations in ligand-gated ion
channels can profoundly affect anesthetic potency
(68,70,74,200). Although the lack of radiograph struc-
tures of ion channels presently thwarts visualization
of anesthetic-induced structural changes, three-
dimensional models of single amino acid mutations in
putative anesthetic targets allow a mechanistic inter-
pretation of the effects of these changes and suggest
new mutations (70,201–205). These models allow vi-
sualization of complex interactions, such as those of
widely separated double mutations (68,70,205), and
732 REVIEW ARTICLE SONNER ET AL.
MECHANISMS UNDERLYING MAC
Figure 11. A molecular model of a transmembrane (TM) segment of
a ␥-aminobutyric acid (GABA)A ␣1 receptor was built by threading
the primary sequence of a GABA receptor onto a template of a
four-helical bundle found in the three-dimensional structure of
cytochrome oxidase (208). The five subunits each are composed of
four ␣ helices. Four subunit helices are rendered as red cylinders
connected by green interhelical loops. Helices show discontinuities
at proline residues. The fifth subunit shows the amino acid back-
bone trace as a yellow ribbon and shows the three amino acid
residues most important for modulation of anesthetic potency: L232,
S270, and A291 (rendered with a space-filling surface in which the
carbon, oxygen, and hydrogen atoms are colored green, red, and
white, respectively). (a) The transmembrane segment of the receptor
from the side in the plane of the membrane and (b) the ion pore of
the receptor looking down the fivefold symmetry axis from the
extracellular side are shown.
thereby suggest far more efficient tests than the pro-
duction of random double mutations.
Secondary structure prediction algorithms indicate
that bundles of four ␣ helices form the subunits of the
transmembrane segment of homopentameric GABAA
receptor ␣1 and glycine receptor ␣1 ion channels (206).
The ␣ helical nature of transmembrane segment 2 in
these ligand-gated ion channels is consistent with ob-
servations of electron density in cryoelectron micro-
graphs of acetylcholine receptors by Unwin et al.
(207), with the crystal structures of a four-␣ helical
bundle found in a bacterial cytochrome (208) and of a
bacterial mechanosensitive ion channel (209). Such
considerations led to models of these channels (and
others in the “Cys-loop” superfamily) (201,204,208).
Cavities in the central axis of these bundles share
many properties, including volume and polarity, with
binding sites in four-helical bundles engineered by
Johansson et al. (210) and Tanner et al. (211). The latter
binding sites bind halothane with Kd values close to
MAC. Combining five of the four ␣-helix subunits
allows formation of a homopentameric ion channel
(Fig. 11). The second transmembrane segments of each
of the five ␣ helices form a central pore with a right-
hand supertwist and a funnel shape that is most nar-
row at the intracellular face (212).
Molecular dynamics simulations of anesthetic mol-
ecules in proteins and phospholipid bilayers may re-
veal how and where anesthetics act because such sim-
ulations can provide insights into anesthetic effects on
the forward and backward rate constants that control
the equilibrium between resting, open, and desensi-
tized states of ion channels (213). Partition coefficient
measurements (214) provide anesthetic distributions
ANESTH ANALG
2003;97:718 –40
within pure compounds, whereas molecular dynamics
simulations reveal anesthetic molecule locations
within the heterogeneous mixtures of protein, lipid,
and water that characterize nerve membranes (215).
Molecular dynamics studies demonstrate that anes-
thetic molecules distribute throughout the phospho-
lipid bilayer with a preferential localization near phos-
pholipid head groups (216,217).
Molecular modeling, combined with x-ray crystal-
lography and nuclear magnetic resonance (NMR)
structure determination, can reveal atomic details of
ion channel structure and function. The structure of a
bacterial mechanosensitive channel provides a tem-
plate for pentameric ion channels (209) that underlies
molecular modeling predictions of the mechanism of
channel opening and open state structure (218). By
using NMR structures of helices in solution (219,220),
several studies modeled the structure of the five heli-
ces surrounding the pore in the acetylcholine receptor
(220,221). The radiograph structure of the potassium
channel (222) permits detailed molecular models of
the ion selectivity filter (223,224).
Convergence of the three applications described
above may indicate whether single amino acid muta-
tions change alcohol-/anesthetic-binding sites or
whether they alter protein stability in a way that al-
lows anesthetic molecules to act remotely by nonspe-
cific mechanisms. At present, considerable evidence
indicates that site-directed mutations modify the vol-
ume and polarity of specific anesthetic binding sites.
Thus, at the level of atomic detail, the binding site in a
GABAA ␣1␤2␥2 receptor will have a different volume
and polarity than a similar site in a GABAA ␣1␤3␥2
receptor. Given sufficient information about the phar-
macophores that determine anesthetic binding in a
particular cavity, it may be possible to design anes-
thetic molecules that bind to the former site, but not to
the latter. Such a “designer” molecule could offer in-
creased selectivity of regions of the nervous system
affected by anesthetics. For example, it might be pos-
sible to target receptors present in the spinal cord, but
not in the hippocampus.
The 5-Angstrom Hypothesis
Results of studies of partially fluorinated series of
n-alkanes and alcohols suggest that inhaled anesthet-
ics may produce immobility by concurrent actions on
two sites separated by 5 angstroms (225). Five ang-
stroms (fortuitously?) approximates the distance that
separates adjacent loops of an ␣ helix (226), or the
distance between some sites on adjacent transmem-
brane segments, or the effective pore diameter of gly-
cine and GABAA receptors (227). The ultimate signif-
icance of this finding remains unclear.
ANESTH ANALG
2003;97:718 –40
Nonspecific Mechanisms
The preceding observations could be interpreted to
indicate that actions on ion channels cannot entirely
explain the capacity of inhaled anesthetics to produce
immobility—although changes in transmission gov-
erned by such channels ultimately must underlie this
capacity. Several lines of evidence support this minor-
ity view. In vitro studies of specific receptors usually
demonstrate wide variations in the potencies of dif-
ferent anesthetics: in vitro potencies do not always
correlate with MAC (e.g., Fig. 5), and, for a given
receptor, different anesthetics may produce opposing
effects (stimulation by some and depression by others)
(113). Present data support a role for glycine and a few
other receptors, but for the most part, such roles are
limited. No inhaled anesthetic action on a single re-
ceptor can explain immobility, and immobility as a
result of concurrent actions on many receptors is un-
likely (228), particularly because several proposed
candidates now seem less likely (e.g., acetylcholine
and GABAA).
The importance of effects on receptors versus some
nonspecific effect may be gauged from Figure 1. Af-
finity to a lipid phase is a measure of a nonspecific
effect. If receptors play a role as mediators, then dif-
ferences in receptor effects (as determined in vitro)
might be reflected in deviations from the correlation
with lipophilicity. However, the data illustrated in
Figure 1 do not demonstrate marked deviations. The
values for cyclopropane and halothane lie equally
close to the line, but these anesthetics differ consider-
ably in their in vitro enhancement of glycine receptors.
Similarly, benzene and hexafluorobenzene differ in
their in vitro capacities to block NMDA receptors
(Raines D, unpublished data) but are approximately
equidistant from the line of correlation.
If immobility does not result from direct actions on
one or many channels, how might it be produced? Have
we prematurely discarded nonspecific mechanisms, par-
ticularly those involving the membrane bilayer? Cantor
(229 –231) suggested that there are enormous counterbal-
ancing pressures (averaging approximately 300 atm)
within the hydrophobic core of the lipid bilayer of the
plasma membrane. These pressures compensate for the
attractive interfacial tensions at the aqueous interfaces of
the membrane. Variations in the distribution of such
pressures, as would accompany the incorporation of gas-
eous or vapor molecules, are predicted to strongly influ-
ence the activity of transmembrane proteins (e.g., ion
channels). The changes in activity might produce anes-
thesia or other CNS effects, such as the convulsions seen
with nonimmobilizers. Although Cantor’s predictions
have not been adequately tested experimentally, there is
some accord with experimental results. The theories pre-
dict sigmoidal ion channel dose-response curves, sug-
gesting marked allosteric effects in the absence of protein
REVIEW ARTICLE SONNER ET AL. 733
MECHANISMS UNDERLYING MAC
binding (232). Also, a redistribution of lateral pressures
within the membrane in response to a solution of volatile
compounds within the membrane can predict the low
potency of some compounds that disobey the Meyer-
Overton rule (229). Reexamination of observations cited
as evidence against indirect mechanisms does not ex-
clude Cantor’s theory. For example, his theory does not
predict that increasing temperature should cause anes-
thesia (229), and small stereoselective effects of anesthet-
ics may be seen with either a protein- or membrane-
based mechanism because both proteins and lipids have
chiral centers.
Using molecular dynamics simulation, Tang and Xu
(233) found that halothane probably does not act on a
hydrophobic protein pocket. They suggested that
global, rather than local, effects underlie the actions of
halothane. Thus, a few investigators still believe that
variants of Meyer’s (4) and Overton’s (3) hypothesis
may be correct.
Conclusions
Our view and knowledge of the mechanisms by which
inhaled anesthetics act to produce immobility have
expanded in the past two decades. Actions on one or
a few spinal cord receptors may underlie immobility.
Afferent (sensory) pathways, efferent (motor) path-
ways, or interneurons might mediate immobility. Be-
cause they provide the final common pathway for
pain-evoked movement, depression of motor neuron
receptors (by pre- and postsynaptic effects on excita-
tory and inhibitory neurotransmission) has received
particular study.
Although results from in vitro studies show that
relevant concentrations of inhaled anesthetics can
modulate ion channels in ways that could cause im-
mobility, only a few plausible candidates may be rel-
evant (Table 3). These include some neurotransmitter-
gated ion channels. Molecular, neurophysiologic, and
behavioral investigations support a role for glycine
receptors (e.g., Fig. 5), but glycinergic effects do not
explain most of the immobilizing action of any inhaled
anesthetic. The putative binding site underlying the
enhancement of glycine receptors by inhaled anesthet-
ics lies near the extracellular membrane interface.
In contrast to evidence supporting glycine receptors
as direct mediators of MAC, other members of the
ligand-gated ion channel superfamily (GABAA, neu-
ronal nACh, and 5-HT3 receptors) probably do not
mediate the immobility produced by inhaled anesthet-
ics, despite compelling evidence that GABAA recep-
tors are responsible for the immobilizing effects of
some injected anesthetics. This does not exclude im-
portant roles for these receptors for other aspects of
anesthesia. Some metabotropic receptors that do not
appear to be important to the immobilizing action of
734 REVIEW ARTICLE SONNER ET AL. ANESTH ANALG
MECHANISMS UNDERLYING MAC 2003;97:718 –40

Table 3. Relevance of Specific Receptors as Direct Mediators of MAC

Receptor Rationale
Relevant
Glycine receptors Spinal cord distribution; anesthetics prolong glycinergic duration of miniature
postsymptic currents at clinically relevant concentrations; effect of antagonists
on MAC correlates with in vitro effect
Possibly relevant
5-HT2A receptors Intrathecal ketanserin decreases MAC by 20%–25%, but no in vitro data support a
direct effect
Sodium channels Systemic lidocaine decreases MAC by 40%–50%; anesthetics can inhibit sodium
channels at relevant concentrations, but nonimmobilizers do not; blockade of
sodium channels may decrease glutamate release from NMDA nerve terminals
NMDA receptors In vitro, several inhaled anesthetics can block NMDA receptors at clinically
relevant concentrations; NMDA receptor blockade decreases MAC, and the
decrease correlates with blocker concentration in the spinal cord; inhaled
anesthetic blockade of sodium channels of nerve terminals may decrease
glutamate release and thus decrease NMDA receptor activity; we speculate that
enhancement of glycine receptors on NMDA-containing nerve terminals may
increase glutamate release and thus decrease NMDA receptor activity; temporal
summation appears to persist during isoflurane but not during xenon
anesthesia; and NMDA blockade eliminates temporal summation for isoflurane
Possibly irrelevant
Potassium channels One knockout does not change MAC; intrathecal riluzole does not decrease MAC
at nontoxic infusions or decreases MAC by the same amount as IV infusions
AMPA and kainate receptors Although AMPA/kainate receptor blockade decreases MAC and anesthetics
inhibit AMPA-/kainate-mediated excitatory postsynaptic currents (but only a
little), GluR2 null mutant mice have normal MAC values
Probably irrelevant
GABAA receptors Increase in MAC from intrathecal administration of GABAA antagonists does not
correlate with in vitro effect; inconsistent relationship of MAC to in vitro effects;
IV administration of antagonists increases MAC as much as ketamine ED50
Opioid receptors Naloxone does not change MAC
␣2 adrenoreceptors Systemic and/or intrathecal blockers and depletors do not appreciably increase
MAC
5HT3 receptors Ondansetron does not affect MAC; some in vitro anesthetic effects are excitatory
Acetylcholine receptors Blockers of nicotinic and muscarinic receptors and combinations of the blockers
do not change MAC; the nonimmobilizer F6 inhibits nicotinic cholinergic
receptors
MAC ⫽ minimum alveolar anesthetic concentration; GABA ⫽ ␥-aminobutyric acid; NMDA ⫽ N-methyl-d-aspartate; AMPA ⫽ ␣-amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid; 5-HT ⫽ 5-hydroxytryptamine; ED50 ⫽ 50% effective dose; F6 ⫽ 1,2-dichlorohexafluorobutane.

inhaled anesthetics include muscarinic acetylcholine
receptors, opioid receptors, and ␣2-adrenergic recep-
tors. 5-HT2A receptors may contribute a small direct
effect, although no direct effects of anesthetics on
5-HT2A receptors have been demonstrated.
NMDA receptors may be candidate targets of vola-
tile anesthetic action. Kainate receptors probably are
not targets because in vitro effects require large anes-
thetic concentrations. The same may be said of AMPA
receptors. The lack of effect of a knockout of an AMPA
receptor GluR2 subunit on MAC adds to this conclu-
sion, but, of itself, such a lack may not be sufficiently
defining, because ventral horn (but not dorsal horn)
neurons lack GluR2.
Blockade of voltage-gated sodium channels by in-
haled anesthetics may produce presynaptic effects
that underlie MAC. Enhancement of potassium chan-
nel function also has not been excluded as a possible
mechanism, although negative studies in one knock-
out animal and with a nonspecific KCNK channel
activator call into question the relevance of these chan-
nels. However, characterization of anesthetic effects
on K channels has been performed for only a few
members of this large family.
Combined pharmacologic and genetic approaches al-
low a determination of the likely relevance of specific ion
channels as mediators of MAC. Several receptors and
ion channels previously thought to be likely candidates
(acetylcholine, GABAA, 5-HT3, ␣2-adrenergic, and
opioid receptors and potassium channels) now seem
less likely (but some may be crucial to other aspects of
anesthesia, such as amnesia); glycine and NMDA recep-
tors and sodium channels seem more likely candidates.
Developments in the next few years should lead to a
definitive understanding of how inhaled anesthetics pro-
duce immobility.
ANESTH ANALG
2003;97:718 –40
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