A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral
components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro
approaches, we found that GABAAreceptors that contain the ?5 subunit (?5GABAARs) play a critical role in amnesia caused by the
edly increased a tonic inhibitory conductance generated by ?5GABAARs, whereas synaptic transmission was only slightly enhanced.
activity were similar in WT and ?5?/? mice. Deletion of the ?5 subunit of the GABAARs reduced the amnestic but not the sedative-
hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of
General anesthetics are highly lipid-soluble, low-potency com-
pounds that were initially thought to act by nonspecifically per-
turbing the structure of lipid bilayers. A major advance in the
1980s was the identification of neuronal proteins as anesthetic
targets (Franks and Lieb, 1988). Behavioral and neuroimaging
studies in humans and animal models have since shown that
anesthetics cause a compilation of different behavioral end-
points, including amnesia, immobility, and unconsciousness,
that are mediated by different brain regions and receptor popu-
and null mutant mice have shown that specific populations of
GABAAreceptors (GABAARs) contribute to the sedative and im-
mobilizing properties of certain anesthetics (for review, see Ru-
dolph and Antkowiak, 2004). The molecular targets underlying
the amnestic properties of anesthetics have been highly elusive
but are of great clinical importance and scientific interest. Unin-
tended intraoperative awareness during surgery occurs in one or
two cases per 1000 anesthetized patients (Sebel et al., 2004). Be-
cause anesthetics are administered to ?27 million patients each
year, intraoperative awareness has become a major medical con-
cern, as highlighted by a “sentinel alert” released by the Joint
Commission on Accreditation of Healthcare Organizations
thetics have not been identified previously.
including etomidate, are positive allosteric modulators of
hippocampus, a brain structure that is involved in learning and
memory, GABAARs generate two distinct forms of inhibition
vesicular release of GABA and the activation of postsynaptic
GABAARs, whereas a low-amplitude tonic inhibitory conduc-
tance is generated by low concentrations of ambient GABA (Se-
myanov et al., 2004). In hippocampal pyramidal neurons,
of Anesthesia, University of California, San Francisco, CA) for performing the HPLC measurements of etomidate
TheJournalofNeuroscience,April5,2006 • 26(14):3713–3720 • 3713
tinct subunit compositions and pharmacological properties
(Yeung et al., 2003; Caraiscos et al., 2004a). We have shown pre-
viously that a tonic inhibitory conductance in hippocampal py-
GABAARs (?5GABAARs) (Caraiscos et al., 2004a). The ?5 sub-
unit is of particular interest in memory processes, because it is
2000). In humans and rodents, only 4% of all GABAARs in the
brain but 25% of GABAARs in the hippocampus contain the ?5
subunit (Sur et al., 1999). Several lines of evidence suggest that
?5GABAARs play a major role in memory processes, because
reduced expression of the ?5 subunit is associated with better
performance of hippocampal-dependent learning tasks (Col-
linson et al., 2002; Crestani et al., 2002). Also, inverse agonists
selective for ?5GABAARs have nootropic effects in animal mod-
els (Chambers et al., 2003). Here, we test the hypothesis that
etomidate, a prototypic intravenous anesthetic, increases the
function of ?5GABAARs and that this effect contributes to the
amnestic but not the sedative-hypnotic properties of this
Generation of ?5 null mutant (?/?) mice and cell cultures. The experi-
Toronto. The generation of the ?5?/? mice has been described previ-
ously (Collinson et al., 2002). Notably, ?5?/? mice exhibit normal life
changes in other GABAAR subunits. Cultures of hippocampal neurons
from ?5?/? mice and wild-type (WT) littermates were prepared as
previously described (MacDonald et al., 1989) on postnatal day 1. For
several experiments, hippocampal cultures were prepared from embry-
onic Swiss White mice because the number of WT mice available from
the heterozygous ?5?/? breeding pairs was limited. To increase the
acterization, cultures of dissociated neurons were treated with the
GABA-transaminase inhibitor vigabatrin (100 ?M), before recording
(Wu et al., 2003), although a low-amplitude tonic current can be readily
detected in cultured hippocampal neurons without such treatment (Bai
et al., 2001).
Electrophysiology. The concentration-dependent effects of etomidate
on tonic and synaptic currents in cultured hippocampal neurons were
determined with the whole-cell patch-clamp technique (at 20–23°C).
Electrodes were made from borosilicate glass pipettes and fire polished
at 10 kHz before digitization (Digidata 1200; Molecular Devices). Series
resistance and pipette and whole-cell capacitance were cancelled elec-
10 mV was applied. Only cells that demonstrated stable series resistance
(?20% change) were used for data analysis. Cells were perfused with a
25 HEPES, and 28 glucose, pH 7.4. Tetrodotoxin (0.3 ?M) was added to
the extracellular solution to inhibit spontaneous voltage-dependent so-
dium channel activity. In all experiments, potassium currents were sup-
pH 7.3, that contained the following (in mM): 120 CsCl, 2.0 MgCl2, 1.0
CaCl2, 11 EGTA, 30 HEPES, 2.0 MgATP, and 2.0 tetraethylammonium.
The amplitude of the tonic current under control conditions was mea-
sured as the difference in the holding current before and during the
application of bicuculline methiodide (100 ?M). Etomidate (Bedford
Laboratories, Bedford, OH) or the vehicle control (35% v/v propylene
glycol) at equivalent concentrations was added to the extracellular solu-
tion. Solutions were applied to the cell cultures using a multibarrel fast
For synaptic currents, peak amplitude, charge transfer [Q, the inte-
grated area under miniature IPSCs (mIPSCs)], and time constant of
a single exponential equation in the form I(t) ? Aoexp (?t/?off) ? C,
where I(t) is the current amplitude at any given time t, C is the residual
current, and Aois the current amplitude at time 0. The change in charge
previously (Bai et al., 2001) using the following equation: ?QmIPSC?
fdrug? Qdrug? fcon? Qcon, where fdrugand fconare the frequencies (in
Hertz) of mIPSCs, Qdrugand Qconare the average charge transfer (pC)
per mIPSC during drug and control conditions, respectively. Under our
experimental conditions, we assumed that the change in charge transfer
reflected a proportional change in membrane conductance. The charge
?QTC? ITC? ?t, where ?QTCis the charge transfer produced by the
tonic current, and ITCis the current amplitude at steady state. In addi-
tional experiments, the long-term potentiation (LTP) of excitatory po-
to 9-month-old male WT and ?5?/? mice. After administration of
halothane anesthesia, mice were decapitated and their brains were
quickly removed and placed in ice-cold oxygenated (95% O2, 5% CO2)
artificial CSF (aCSF) (composition in mM: 124 NaCl, 3 KCl, 1.3 MgCl2,
2.6 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 D-glucose) with the osmolar-
ity adjusted to 300–310 mOsm. Slices (350 ?m thick) containing trans-
verse sections of the hippocampus were prepared with a vibratome
in the oxygenated aCSF, slices were transferred to a submersion-
a surgical cut, and slices were continually perfused with aCSF. Extracel-
neurons using electrodes that contained aCSF. Synaptic responses were
concentric bipolar stimulating electrodes (Rhodes Medical Instruments,
Summerland, CA). Baseline responses were obtained by stimulation at
0.05 Hz using an intensity that yielded a half-maximal field potential
slope. LTP was induced by one episode of theta-burst stimulation (TBS)
at the same intensity as that used for the baseline response. The TBS
protocol consisted of 10 stimulus trains delivered at 5 Hz, each train
consisting of four pulses at 100 Hz. TBS was induced after the slice had
higher concentration of etomidate was used for the hippocampal slices
than for the neuronal cultures, because slow diffusion and equilibration
means that higher concentrations of lipophilic anesthetics are needed to
dell et al., 2004).
Behavioral tests. All behavioral tests were performed using male age-
matched ?5?/? and WT mice. The experimenters were blind to mouse
genotype and drug treatment.
Perspex arena with a light mounted in the lid (350 ? 200 ? 193 mm;
Technical and Scientific Equipment, Midland, MI). The floor consisted
of stainless steel bars (4 mm diameter, 5 mm apart) that were connected
to a computer, which controlled the duration of the test session and the
timing, intensity, and duration of the shock. On day 1, single subjects
were allowed to explore the chamber for 180 s. They then received three
the drug studies, etomidate (4 mg/kg) or vehicle (propylene glycol in
sterile saline at concentration and dose volume equivalent to the
etomidate-containing solution) was administered intraperitoneally be-
fore the conditioning trial. On day 2, 24 h after the conditioning session,
mice were returned to the chamber, and the freezing response was as-
sessed immediately then every 8 s for 8 min. The freezing response was
defined as the lack of any movement except that required for breathing.
Assessment of the freezing response occurred in the same conditioning
chamber in which the mice received the foot shocks.
Morris water maze. For the Morris water maze probe trial, a circular
opaque by the addition of a white nontoxic paint. A circular platform
with a diameter of 12 cm was placed ?0.5 cm below the water surface so
trials each day. In the matching-to-place water maze, the location of the
3714 • J.Neurosci.,April5,2006 • 26(14):3713–3720Chengetal.•MolecularSubstratesofAnesthetic-InducedAmnesia
The ?5?/? mice used in this study have been reported previously to
exhibit better performance in the initial acquisition trials of the Morris
water maze paradigm (Collinson et al., 2002). We initially tested the
effects of etomidate on performance during the acquisition trials in WT
and ?5?/? mice; however, the results were extremely variable, so this
undertaken to test for spatial learning. During the acquisition phase of
the probe trials, each mouse was randomly assigned to receive an injec-
Bimeda-MTC Animal Health, Cambridge, Ontario, Canada). Thirty
minutes later, the mouse was placed in the water maze. Four acquisition
trials were conducted after the injection. The next day, a probe trial was
performed to test the ability of the mice to recall the spatial location that
previously contained the hidden platform. During the probe trial, the
to search for 60 s. The mouse was then promptly rescued, and the trial
ended. Mice that learned the correct location of the platform selectively
searched in the correct area. The percentage of time spent within the
correct area of the pool (four times the diameter of the platform) was
used to measure recall. The correct location of the platform represented
16% of the total area of the pool. If no learning occurred, the mouse was
expected to spend 16% of the time near the correct location. The vehicle
and drugs were both administered twice, each on separate days. The
order in which the drug or vehicle was administered was random. The
results from the two probe trials were averaged to provide a single value
of the vehicle control. Data records were made with HVS Water 2020
software (VHS Image, Hampton, UK) for off-line analysis. Briefly, a
video camera captured the movement of the mouse, and HVS Water
path, and latency of each mouse during a trial were recorded, and the
percentage of time spent in the correct region was calculated by the
software during analysis.
Rotarod. Mice were tested on a rotating rod unit (rotarod) to study
motor coordination and strength. The mice were trained to walk on a
rotarod (Economex; Columbus Instruments, Columbus, OH) revolving
at a constant speed of 12 rpm for 120 s consistently. On the test day, one
preinjection trial was performed before the animals were treated with
vehicle or etomidate. Performance was indicated by the latency to fall
remained on the rotarod was recorded up to a maximum of 120 s.
Open field test. The sedative properties of etomidate were tested by
for five consecutive minutes. After placement in the open field, a trained
examiner used an event recorder to score the total time spent walking.
of the test chamber with a mild ethanol solution between tests. A heat
lamp was placed directly above the open field to accommodate for the
hypothermic effects of etomidate.
Loss of righting reflex. The loss of righting reflex (LORR) was assessed
using a classical experimental protocol. The LORR was determined in
WT and ?5?/? mice for a wide range of etomidate doses (5–20 mg/kg,
back and scored as anesthetized if they failed to completely right within
they were scored as awake. All mice were used only once for the LORR
at each dose of etomidate. To determine the dose of etomidate that
fit, using nonlinear regression, to the equation: Y ? D ? [A ? D]/[1 ?
10((logED50-X) Hill slope)],whereDistheminimumresponse,Aisthemax-
imum response, and X is the dose. The time to the LORR was also quan-
tified by measuring the time from the end of the injection to when the
mice first demonstrated a LORR.
Brain concentration of etomidate. HPLC was used to measure the con-
centration of etomidate in the brains of the mice. The measurements
were performed according to a method reported previously (McIntosh
and Rajewski, 2001). The brains were collected, weighed, homogenized,
and frozen at ?80°C. An HPLC unit (Hewlett-Packard 1100 series; Agi-
lent Technologies, Mountain View, CA) was equipped with a variable-
wavelength detector. Etomidate analysis was performed using a reverse-
room temperature. Standard curves were created by adding known
the concentration. The detection wavelength was set at 242 nm. An iso-
cratic mobile phase consisting of 25:25:50 (v/v/v) of acetonitrile, metha-
nol, and 25 mM phosphate buffer, pH 8.1, was used at a flow rate of 1.5
Statistics. All results are reported as the mean ? SEM. Statistical sig-
nificance was assessed with the Student’s t test or one-way or two-way
ANOVA and the Newman–Keul or Bonferroni’s post hoc tests, as
Etomidate enhanced tonic conductance in pyramidal neurons
The concentration-dependent effects of etomidate on tonic and
synaptic currents were first studied using whole-cell currents re-
corded in pyramidal neurons grown in dissociated cultures. The
cell culture preparation permits more effective control of drug
is possible with brain slices. The application of a low concentra-
tion of etomidate (0.1 ?M) to pyramidal neurons from Swiss
(156 ? 24% of control; n ? 8; p ? 0.05), whereas etomidate (0.1
?M) failed to alter the time course or amplitude of mIPSCs (Fig.
the inhibitory net charge transfer was 60-fold greater for tonic
conductance than for synaptic conductance. This indicated that
on tonic conductance than on synaptic conductance under the
experimental conditions (Fig. 1B).
tance was generated by ?5GABAARs, currents were recorded
from neurons obtained from genetically modified mice that
etomidate would minimally increase the holding current in
Chengetal.•MolecularSubstratesofAnesthetic-InducedAmnesiaJ.Neurosci.,April5,2006 • 26(14):3713–3720 • 3715
?5?/? neurons relative to WT neurons,
whereas higher concentrations of etomi-
date would cause similar enhancement of
synaptic currents in the two groups. The
the percentage change in the tonic cur-
rent was minimal in ?5?/? neurons
(Caraiscos et al., 2004a). These studies were
was a compensatory upregulation of other
high-affinity GABAARs, which might be
in ?5?/? neurons and whether there were
differences in the etomidate sensitivity of
synaptic currents in WT and ?5?/?
rons (29.7 ? 13.9 pA; n ? 7) but not ?5?/? neurons (4.2 ? 2.2
pA; n ? 7) ( p ? 0.05) (Fig. 2A,B). No differences were detected
in the frequency, amplitude, or time course of mIPSCs between
(0.1 ?M) (Table 1). Thus, low concentrations of etomidate that
are reported to be clinically relevant (0.05–0.43 ?M) (Rudolph
current than the mIPSCs. Higher concentrations of etomidate
caused a further increase in the holding current and prolonged
criminate effect of etomidate on GABAARs that do not contain
the ?5 subunit.
Etomidate reduced LTP in WT but not ?5?/? brain slices
excitatory synapses. LTP is widely regarded as a possible cellular
model of learning and memory (Bliss and Collingridge, 1993;
GABAAR antagonists enhance LTP, whereas positive allosteric
modulators reduce LTP and impair memory (Seabrook et al.,
1997). Our aim was to determine whether etomidate differen-
tially modulated the LTP of fEPSPs recorded at CA1 pyramidal
neurons from WT and ?5?/? mice.
As reported previously (Collinson et al., 2002), no differences
in LTP were observed between vehicle-treated WT and ?5?/?
per condition; p ? 0.05) or 60 min after TBS (170 ? 15 vs 175 ?
11%; n ? 6; p ? 0.05) (Fig. 3A,B). Etomidate-treated (1 ?M)
fEPSPs (178 ? 16% of control; n ? 6 slices) immediately after
fEPSPs decreased to 103 ? 13% of control at 60 min (n ? 6; p ?
0.05). Similarly, the LTP of fEPSPs was initially observed in
(Fig. 3A,B) immediately after TBS. Unlike the recordings in WT
slices, enhancement of the slope of the fEPSPs in ?5?/? slices
was sustained at 60 min after TBS (168 ? 14% of control; n ? 6;
attenuation of LTP by etomidate was absent in slices from
We also measured the amplitude of the tonic current gener-
ated by etomidate (1 ?M) in CA1 pyramidal neurons in hip-
pocampal slices under voltage-clamp conditions. As observed in
recordings from cultured hippocampal neurons, etomidate
caused a multiple-fold greater increase in the tonic current in
CA1 pyramidal neurons from the WT mice, compared with
?5?/? mice (26.4 ? 4.9 pA, n ? 10 vs 3.4 ? 1.6 pA, n ? 10,
respectively; p ? 0.05).
Amnestic effects of etomidate are mediated by ?5GABAARs
The electrophysiological experiments showed that low concen-
trations of etomidate enhanced the tonic current and reduced
LTP in pyramidal neurons from WT but not ?5/? mice. These
attenuated in ?5?/? mice. To study the effects of etomidate on
hippocampal-dependent memory, two complementary behav-
ioral assays were performed, a contextual fear conditioning par-
adigm and the matching-to-place version of the Morris water
Contextual fear conditioning was used to measure the ability
of the mice to learn and remember an association between an
adverse experience and environmental cues (Fanselow, 1980).
WT and ?5?/? mice treated with the vehicle exhibited similar
freezing behavior (83.1 ? 3.7% of time spent freezing, n ? 8 vs
mg/kg, i.p.) reduced the freezing scores in WT but not ?5 ?/?
mice (52.7 ? 5.0%, n ? 8 vs 78.8 ? 5.1%, n ? 9; p ? 0.05) (Fig.
4A). These findings indicate that etomidate impaired the acqui-
sition of contextual fear in WT but not ?5?/? mice. To ensure
that the reduced etomidate sensitivity exhibited by the ?5?/?
mice was not a result of a nonspecific insensitivity to general
anesthetics, the effect of another anesthetic that does not target
GABAARs was tested. The dissociative anesthetic ketamine is
?5?/? neurons. A, Current traces illustrate the increase in an inward current at different
the holding current was greater in WT than in ?5?/? neurons. Note that the scale of the
Low concentrations of etomidate increased the holding current in WT but not
3716 • J.Neurosci.,April5,2006 • 26(14):3713–3720 Chengetal.•MolecularSubstratesofAnesthetic-InducedAmnesia
thought to cause amnesia by inhibiting the NMDA subtype of
glutamate receptors. As shown previously, the vehicle had no
effect in WT and ?5?/? mice (76.9 ? 5.5% of time spent in
ketamine (20 mg/kg, i.p.) caused impairment in both WT and
?5?/? mice, as shown by a similar reduction in freezing scores
(WT mice, 34.8 ? 8.7% of the time, n ? 8, p ? 0.05; ?5?/?
mice, 35.8 ? 5.7% of the time, n ? 8, p ? 0.05) (Fig. 4A,B).
The Morris water maze was next used as an independent test
of spatial learning. Etomidate (4 mg/kg, i.p.) impaired memory
time that mice spent swimming in the area that had previously
contained the hidden platform (control, 21.2 ? 2.6% of time
spent in the correct location vs etomidate, 14.0 ? 1.6%, n ? 16;
p ? 0.05) (Fig. 4C). The reduction in performance was not ex-
hibited by the etomidate-treated ?5?/? mice (control, 21.7 ?
also tested the effects of ketamine on the ability of the mice to
locate the platform during a probe trial. Ketamine (20 mg/kg,
i.p.) caused a similar impairment of spatial learning in WT mice
(control, 22.1 ? 2.7% vs ketamine, 15.4 ? 2.2%, n ? 17; p ?
0.05) and ?5?/? mice (control, 21.5 ? 2.1 vs 15.0 ? 1.8%; n ?
15; p ? 0.05) (Fig. 4D).
Visible platform trials were performed to test for possible ge-
abilities, and nonspecific effects of etomidate. The platform that
was usually submerged was raised to 0.5 cm above the waterline
and marked with a brightly colored flag. The mice were injected
platform trial. There were no differences in the latency to locate
the platform between the two genotypes, in the absence or pres-
ence of etomidate (WT control, 5.4 ? 0.8 s, n ? 8 vs WT etomi-
8 vs ?5?/? etomidate, 5.5 ? 0.8 s, n ? 8, p ? 0.05) (Fig. 4E).
Also, no difference in mean swimming speed was detected be-
control, 0.23 ? 0.01 m/s, n ? 16 vs WT etomidate, 0.22 ? 0.01
trials (Fig. 4F). The lack of difference in swim speed (in the ab-
sence or presence of etomidate) for both
groups confirmed the procedural ability
of the mice to perform the task.
Sedative-hypnotic effects of etomidate are
not mediated by ?5GABAARs
Performance on the rotarod measures
sensorimotor control and coordination
and is thought to depend primarily on
neuronal circuits in the cerebellum and
spinal cord. To ensure there were no dif-
ferences in sensorimotor function in WT
and ?5?/? mice at the dose of etomidate
and time points that were selected for the
behavioral experiments, we examined im-
pairment of walking on the rotarod. Five
minutes after the injection of etomidate,
No impairment of performance was de-
tected 30 min after etomidate (4 mg/kg,
i.p.), which was the dose selected for the
fear conditioning and Morris water maze
and the LORR, respectively. These behavioral endpoints were
(Nelson et al., 2002). The sedative properties of etomidate were
studied with an open field test, which examines spontaneous lo-
comotor activity after injection of etomidate or vehicle control.
vehicle (Fig. 5B). Etomidate (4 and 10 mg/kg) caused a similar
reduction in spontaneous movement at 30 min in WT and
?5?/? mice as shown in Figure 5B.
anesthetic-induced unconsciousness is the LORR (Rudolph and
Antkowiak, 2004). The ability of a wide range of etomidate doses
to cause the LORR was determined for WT and ?5?/? mice. A
dose–response plot for LORR showed that the dose of etomidate
that caused half the maximal response was similar in WT and
0.05). The time to LORR was also measured after injection of the
the WT and ?5?/? mice. Together, the above results show that
the ?5?/? mice do not exhibit a resistance to etomidate for the
behavioral endpoints used to measure sedation and loss of
The brain concentration of etomidate was similar in WT and
To determine whether differences in the absorption or metabo-
amnestic effect of etomidate, the brain concentrations of etomi-
date were measured using HPLC. Etomidate (4 mg/kg, i.p.) was
min) as for the fear conditioning and Morris water maze experi-
0.79 ? 0.33 ?M, n ? 4; p ? 0.05). No etomidate was detected in
Thus, differences in the behavioral performance of WT and
?5?/? mice could not be attributed to a difference in the brain
Chengetal.•MolecularSubstratesofAnesthetic-InducedAmnesiaJ.Neurosci.,April5,2006 • 26(14):3713–3720 • 3717
midate that caused amnesia in mice in vivo was similar to the
etomidate concentration that preferentially enhanced the tonic
conductance in vitro and reduced LTP (Fig. 1B,C).
concentrations of etomidate were similar in WT and ?5?/?
performance, because deletion of the ?5 subunit did not influ-
ence the effect of etomidate on the LORR or spontaneous motor
ing, perception, and motivation were similar in WT and ?5?/?
mice, in the absence and presence of etomidate, as evidenced by
the rotarod and open field tests. These results provide evidence
for a selective population of GABAARs that contributes to the
The findings support the hypothesis that etomidate acts on dif-
ferent GABAAR subtypes, located in different neuronal circuits,
et al., 1997).
Electrophysiological studies showed that a low concentration
dal neurons, whereas higher etomidate concentrations further
increased the tonic conductance and prolonged the duration of
mIPSCs in WT and ?5?/? neurons. The free aqueous concen-
tration of etomidate associated with amnesia in the mammalian
suggest amnesia occurs at ?20–40% of the immobilizing con-
centration of anesthetics (Dwyer et al., 1992). The brain concen-
dose and time point selected for the Morris water maze acquisi-
same context. C, Spatial learning was impaired by etomidate (4 mg/kg, i.p.) in WT but not
day. Impaired memory retrieval was shown by WT but not ?5?/? mice. D, In contrast,
tected in the visible platform trial (E) or swim speed (F) between the two genotypes (WT
control, 0.24 ? 0.01 m/s, n ? 16 vs WT etomidate, 0.24 ? 0.01 m/s, n ? 15, p ? 0.05;
expressed as the latency to fall off the rotarod (mean ? SEM). Both groups had impaired
responses 5 min after injection (*p ? 0.05, one-way ANOVA) but not at 30 or 60 min. B,
Spontaneous locomotor activity (walking) was reduced by etomidate as shown for the open
dependent reduction in locomotion that was similar in WT and ?5?/? mice. C, Dose–
response analysis for etomidate causing the LORR is shown. The data points represent the
parameter h ? 5.8 ? 1.8; ?5?/? ED50? 9.2 ? 1.1, h ? 5.7 ? 1.5, p ? 0.05. D, The
Etomidate impairment of motor coordination, spontaneous motor activity, and
3718 • J.Neurosci.,April5,2006 • 26(14):3713–3720 Chengetal.•MolecularSubstratesofAnesthetic-InducedAmnesia
tion trial and fear conditioning, was 0.77 ?M. A similar concen-
tration of etomidate predominantly enhanced the tonic inhibi-
tory conductance in vitro.
The above results are consistent with our previous report that
showed that low concentrations of the inhaled anesthetic isoflu-
cultured hippocampal neurons (Caraiscos et al., 2004b). Isoflu-
an inhaled anesthetic in the CNS, which causes specific behav-
ioral effects, can be relatively accurately determined (Hemmings
et al., 2005). Inhaled anesthetics readily diffuse from the alveoli
into the capillary blood and equilibrate such that, at steady state,
the concentration in the end-expired gas would be equivalent to
the concentration in the CNS. Thus, the concentration of the
inhaled anesthetic in the brain can be accurately estimated by
measuring the concentration in the exhaled gas. Results from a
previous in vitro study (Caraiscos et al., 2004b) stimulated the
hypothesis that potentiation of ?5GABAAR contributes to the
amnestic properties of anesthetics. In the present study, we used
both electrophysiological and behavioral assays and selected an
injectable anesthetic rather than isoflurane because of the obvi-
havioral testing. This is particularly true for the Morris water
maze, a paradigm that is widely used for investigating the phar-
activation of ?5GABAAreceptors contributes to the amnestic
effects of etomidate. Moreover, the amnestic and sedative-
the pharmacologic properties of the GABAAreceptor subunits.
To further examine the cellular mechanisms underlying
memory impairment by etomidate, EPSPs were recorded in CA1
stratum radiatum. In the absence of etomidate, pyramidal neu-
rons in hippocampal slices showed no differences in the induc-
tion or maintenance of LTP in WT relative to ?5?/? mice as
reported previously (Collinson et al., 2002). The induction of
similar in WT and ?5?/? mice (Collinson et al., 2002). The
authors also reported no differences in the ability of low-
frequency stimuli to activate fEPSPs in CA1 or the dentate gyrus;
however, the ability of paired-pulse stimuli to facilitate the am-
plitude of synaptic potentials was significantly increased in
?5?/? mice. Increased paired-pulse facilitation was specific to
?5GABAARs are not highly expressed. Our results showed that
in WT but not ?5?/? slices, an observation that supports the
hypothesis that LTP is necessary for learning and memory. The
molecular mechanisms underlying this altered excitability by
gic compounds, including benzodiazepines, will be the subjects
of future studies.
Genetic, pharmacological, and electrophysiological studies
using mouse models have previously implicated ?5GABAARs in
learning and memory processes. A knock-in strain of transgenic
mice that expressed an ?5 subunit point mutation (H105R)
showed an unexpected reduction in ?5GABAAR expression in
pyramidal neuronsand improved
hippocampal-dependent learning tasks (Crestani et al., 2002).
Trace fear conditioning but not delayed conditioning or contex-
tual conditioning was facilitated in the ?5(H105R) mice. The
?5?/? null mutant mice used in this study have been reported
previously to exhibit improved performance in the initial acqui-
ered the true criterion for the acquisition of the Morris water
maze task, showed a consistent difference between genotypes
only in the presence of etomidate. The normal performance of
the mice in the visible platform task and their impaired perfor-
mance in the hidden platform task are highly consistent with a
deficit in learning and memory caused by etomidate in the WT
but not ?5?/? mice. To complement the Morris water maze
studies, we also examined fear conditioning. We and others re-
conditioning (Collinson et al., 2002). However, a reduction in
contextual fear was evident after administration of etomidate in
WT mice but not ?5?/? mice. Contextual fear was reduced
similarly in both groups by ketamine, which indicates that the
resistance of ?5?/? mice to etomidate cannot be attributed to a
general resistance to neurodepressive drugs.
The results reported here have implications for human mem-
ory processes. In humans and rodents, ?5GABAARs exhibit sim-
ilar structural, kinetic, and pharmacological properties and sim-
1997; Sur et al., 1999; Wainwright et al., 2000). Compounds that
rently being investigated in humans as potentially nootropic
compounds (Chambers et al., 2003). Our findings suggest that
drugs that selectively increase the function of ?5GABAARs may
also have a therapeutic role. There is a need for drugs that cause
amnesia without sedation or unconsciousness. Such compounds
may be of value for patients whose condition is too unstable to
allow adequate doses of currently available anesthetics or to pre-
vent the formation of unpleasant memories. Furthermore, our
results raise the intriguing possibility of a genetic basis for some
is associated with lack of movement (Sandin et al., 2000). Poly-
morphisms occurring for the human ?5 subunits are associated
with a reduction in receptor function (Papadimitriou et al.,
2001a,b). Moreover, the ?5 subunit is downregulated under cer-
tain pathological conditions, including epilepsy (Houser and Es-
may predispose patients to intraoperative awareness. Alterna-
tively, it is possible that anesthetic effects on ?5GABAARs con-
tribute to the postoperative cognitive and memory dysfunction
that occurs in ?25% of elderly patients (Moller et al., 1998).
Animal studies have shown that subtle long-term deficits in
(Culley et al., 2003, 2004). Drugs that selectively reduce
that occur after general anesthesia.
In conclusion, mice with a null mutation of the ?5 subunit of
the GABAAR exhibited reduced sensitivity to the amnestic but
not the sedative-hypnotic effects of etomidate. Amnesia was
likely caused by a direct interaction between etomidate and
and WT mice. The results contribute growing evidence that
?5GABAARs play a central role in learning and memory pro-
cesses. Additionally, increased tonic inhibition generated by
?5GABAAR may contribute to amnesia caused by GABAergic
Chengetal.•MolecularSubstratesofAnesthetic-InducedAmnesiaJ.Neurosci.,April5,2006 • 26(14):3713–3720 • 3719
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