Journal of Behavioral and Brain Science, 2011, 1, 188-193
doi:10.4236/jbbs.2011.13025 Published Online August 2011 (http://www.SciRP.org/journal/jbbs)
Blockade of Brain γ-Aminobutyric Acid A Receptors
Antagonizes Hypnotic Action of Isoflurane in Rats*
―GABAA Receptor and Isoflurane Induced Hypnosis
Ming Xiong#, Ralph L. da Graca#, Jing Li, Harshitha Kota, Jiang-Hong Ye†
Department of Anesthesiology, University of Medicine and Dentistry of New Jersey,
New Jersey Medical School, Newark, USA
[e-mail], †
Received May 23, 2011; revised July 19, 2011; accepted July 28, 2011
Abstract
Although in vitro studies have demonstrated that isoflurane potentiates the function of γ-aminobutyric acid A
receptors (GABAARs), the in vivo data are controversial. To determine if GABAARs contribute to the
loss-of righting reflex (LORR) induced by isoflurane, we studied the LORR in the absence and presence of
gabazine, a competitive GABAAR antagonist, in Sprague-Dawley rats anesthetized with either isoflurane or
ketamine. Administration of isoflurane and ketamine induced LORR in a dose-dependent manner. Gabazine
significantly antagonized the effect of isoflurane and shifted the dose response curve to the right. In addition,
gabazine prolonged the onset time of LORR induced by isoflurane. Ketamine induced LORR was not af-
fected by gabazine. This indicates that centrally administered gabazine selectively blocks the effect of
isoflurane, and the effect of gabazine is not due to a non-specific CNS excitatory action. These results sug-
gest that the hypnotic effect of isoflurane is at least in part mediated by GABAARs.
Keywords: GABAA Receptors, Gabazine, General Anesthetics, Ketamine, Loss-of Righting Reflex
1. Introduction
A large body of work has attempted to identify the re-
ceptors that mediate the various behavioral components
of the anesthetic state, including amnesia, immobility,
and unconsciousness. In vitro evidence suggests that
ligand-gated ion channels are the major targets of general
anesthetics. γ-aminobutyric acid (GABA) is a major in-
hibitory neurotransmitter in the central nervous system
(CNS). GABAA-receptors (GABAARs) belong to the
ligand-gated channel super family and are involved in
regulation of vigilance, anxiety, muscle tension and me-
mory performance [1,2]. Despite in vitro evidence that
many general anesthetics including isoflurane act on
GABAARs, the relationship of isoflurane-induced hyp-
nosis and the function of GABAARs remains unclear. A
previous animal study has found that isoflurane-induced
loss of consciousness, altered cortical excitability, mus-
cular hypotonia, and decreased respiratory rate were
associated with changes in pontine reticular formation
GABA levels [3]. A more recent animal study has found
that an isoflurane interaction at α5GABAARs contributes
to memory impairment during the early postanesthesia
period [4]. More relevant to current study is a previous in
vivo study in mice found that intraperitoneal injection of
bicuculline, a GABAAR antagonist, had no effect on the
onset time of loss of righting reflex (LORR) induced by
isoflurane [5]. The aim of this present study was to re-
examine if GABAARs contribute to isoflurane induced
loss of consciousness.
2. Materials and Methods
2.1. Animals
Female adult Sprague-Dawley rats (n = 64, Taconic Farms,
NY) weighing 250 - 280 g were studied. Rats were
*This work is made possible by Foundation of University of Medicine
and Dentistry of New Jersey, Newark, NJ; National Institute of Health,
Bethesda, MD, grants: AA015925, AT 001182 and AA016964. Parts o
f
the contents have been presented at meetings: Annual meeting, American
Society of Anesthesiology, October 13, 2007, San Francisco, CA.
#These authors contribute equally to this work.
†Corresponding author.

M. XIONG ET AL.
189
housed individually in polycarbonate cages in ventilation
racks. Animals received food and water ad libitum. The
room was illuminated on a 12 hr light/dark schedule with
lights on at 7:00 AM. All experiments were approved by
the Institutional Animal Care and Use Committee (IA-
CUC) of the University of Medicine and Dentistry of
New Jersey, and conducted according to specifications of
the NIH as outlined in the Guide for the Care and Use of
Laboratory Animals.
2.2. Stereotaxic Surgery
After acclimating to the homecage environment for 1
week, rats were anesthetized with ketamine/xylazine (80
mg/5 mg/kg, i.p.) and a stainless steel single guide can-
nulae (23 gauge; Plastics One, Roanoke, VA) was im-
planted 1 mm above the right lateral ventricle of the
brain. Stereotaxic coordinates were determined from the
rat brain atlas by Paxinos and Watson [6], and positioned
as follows: 1.5 mm lateral to midline, 1.0 mm caudal to
bregma, and 3.0 mm below the skull surface. Cannulae
were affixed with dental resin (orthodontic resin, Caulk
Company, Mitford, DE). Ten days of postoperative re-
covery was allowed before initiating the experimental
trials. Note that, we injected gabazine into the right in-
tracerebroventricular (ICV), instead of both sides due to
the technical difficulty.
2.3. Drugs and Drug Administration
Gabazine (SR-95531) (Sigma Chemical Company, St.
Louis, MO) was dissolved in sterile saline and adminis-
tered through a 28-gauge needle injector (Plastics One)
that passes via the stainless steel guide cannula. The in-
jector was connected to a Hamilton 1.0 µl syringe driven
by a syringe pump (Harvard Instruments, South Natick,
Mass., USA). Saline or a drug dose in a total volume of
0.2 µl was infused over 1 minute into the right lateral ven-
tricle of gently restrained rats via the 28-gauge needle
extending 1.0 mm beyond the guide cannulae tip. Rats
were observed for 10 minutes after finishing injection to
make sure there were no apparent behavioral changes.
Rats were then exposed to general anesthesia induced by
isoflurane or ketamine.
To select the optimal concentration of gabazine, in pi-
lot study, we compared the effects on LORR of a range
of concentrations (14, 41, 68, 136, 204 and 272 µM) of
gabazine with saline. At concentrations ≤ 5 µM gabazine,
no significant difference was observed. At concentrations
≥ 204 µM gabazine induced convulsion in some rats.
Therefore we choose 136 µM gabazine in the experi-
ments.
Assessment of Hypnosis. The primary endpoint for
evaluation of the hypnotic state was the LORR, which
was defined as the inability of animals to right themselves
when positioned in a supine position. The concentrations
of anesthetics necessary for loss of consciousness in hu-
mans are similar to those needed to induce LORR in
animals [1,2,7,8].
Experiment 1. Effect of intracerebroventricular (ICV)
administration of gabazine on the LORR induced by
isoflurane. Thirty-eight animals were used in this test.
Isoflurane (Baxter, Deerfield, IL) was administered using
a Dräger Vaporizer (Isoflurane Vapor 19.1, Drägerwerk
AG, Lübeck, Germany) connected to the O2 supply lines
to the sealed chamber and monitored by Datex-Ohmeda
Capnomac Ultima Anesthesia Monitor, for testing LORR
induced by isoflurane. On the test day, rats were first
randomly divided into two groups: 20 for drug treatment
group (Gabazine injection) and 18 for control group (Sa-
line injection). Each group was further divided into 4 sub
groups randomly (n = 5 in drug treatment group, and n =
4 or 5 in control group) to receive systematic administra-
tion of isoflurane at concentrations of 0.3%, 0.6%, 0.9%,
and 1.2%.
Ten min after ICV administration of gabazine or nor-
mal saline (control), rats were placed in the air-tight cham-
ber, and isoflurane was started to be administered and the
time was counted as 0. When rats appeared sedated from
isoflurane, the chamber was then rocked 30 degrees at 1
minute intervals until evidence of LORR. The rock turned
the rats on their back. If animals did not regain posture
within 10 seconds, LORR was recorded. The estimation
of LORR was made by observers who did not know what
prior drug treatment the animals received. Righting re-
flex was considered restored when animals first regained
an upright position, standing on their feet. Both duration
and percentage of animals exhibiting LORR were mea-
sured.
After the test day, animals were recovered from anes-
thesia and rested for 72 hours. The same experiment was
repeated with random assignment of rats as described
above.
Experiment 2. Effect of ICV administration of gaba-
zine on the LORR induced by ketamine.
Twenty four rats were used in this test. After 7 days of
recovery from cannulation surgery, rats were randomly
divided into 2 groups. Twelve rats received ICV gaba-
zine and 12 received ICV saline. Ketamine was adminis-
tered via intra-peritoneal (IP) injection. Each group was
further divided into 4 sub-groups (n = 3) to receive in-
traperitoneal (IP) ketamine (Hospira, Inc, Lake Forest, IL)
injection at 20, 30, 40, 60 mg/kg. LORR (onset and dura-
tion) was recorded after ketamine IP injection. After this
experiment animals were rested for 72 hours. The same
experiment was repeated two additional times with ran-

M. XIONG ET AL.
190
dom assignment to groups.
Histological verification of cannulae placements. After
completion of the experimental procedures, rats were
overdosed with pentobarbital and decapitated. Brains were
carefully removed and fixed in 4% paraformaldehyde for
4 hours. Coronal brain slices (40 µm) across the cannula-
tion site were prepared using a Zeiss Microm HM 550
Cryostat to confirm cannulae tip in the right ventricule
under microscopy. Animals with cannulae tip located
outside the ventricle were indentified and excluded from
data analysis.
Statistical Analysis. Values are expressed as mean ±
S.E.M. Data were analyzed using paired t test. Data in-
volving multiple comparisons were analyzed with two-
way ANOVA. Values of p < 0.05 were considered sig-
nificant. Dose-response data were fitted as previously
described [2,7] to a logistic equation of the form: P =
100Dn/(Dn + (ED50)n) where P is the percent of the po-
pulation anesthetized, D is the drug dose, n is the slope
parameter, and ED50 is the drug dose for a half-maximal
effect.
3. Results
3.1. Isoflurane Concentrations in the Sealed
Chamber
First we tested the reliability of peak and steady state
concentrations of isoflurane in the sealed chamber. As
indicated in Figure 1, the administration of isoflurane
rapidly increased the concentration of isoflurane in the
chamber and reached the plateau. The time required to
reach the plateau decreased with increasing concentrations
of isoflurane. The average time to peak (desired concen-
tration) was approximately 2 minutes, which followed
with a steady state that remained stable for more than 10
minutes (Figure 1). At 10 min, the test was terminated
by evacuating the isoflurane with a vacuum.
3.2. Gabazine Attenuates the LORR Induced by
Isoflurane
Next, we tested the effects of ICV administration of ga-
bazine on the hypnotic effect of isoflurane. The LORR
was used as the primary measure for isoflurane-induced
hypnosis because it has been reported that the concentra-
tions necessary to produce the hypnotic state in humans
are similar to those needed to induce LORR in animals
[1,2,8]. Seven (out of 38) rats in this experiment showed
cannulae placements outside the right lateral ventricle.
These animals were excluded from the data analysis.
As indicated in Figure 2, in rats who received ICV sa-
line, isoflurane induced LORR in a dose-dependent
Figure 1. The time to peak of isoflurane decreases with in-
creasing isoflurane. Average time to peak was ~2 minutes,
with steady state maintained >10 minutes before vacuum
was applied.
fashion. In rats who received ICV gabazine, a significant
reduction in the percent of rats exhibiting LORR was
observed at both 0.6% and 0.9% isoflurane. This effect
resulted in both a significant rightward shift of the dose
response curve and an increased IC50 value of isoflurane
(from 0.59% ± 0.04% in saline to 0.78% ± 0.03% in
gabazine) (Figure 2(a)). In addition, in rats who received
ICV gabazine and displayed LORR, a significant delay
of onset time of LORR was observed at 0.6% isoflurane
(p = 0.011) and 0.9 % isoflurane (p = 0.049) compared
with rats who received ICV saline (Figure 2(b)).
3.3. Gabazine Does Not Affect the LORR
Induced by Intraperitoneal (IP) Ketamine
Ketamine induces hypnosis via inhibition of NMDA
subtype glutamate receptors in the brain [10]. To confirm
that gabazine reduction of isoflurane-induced LORR is
not due to a non-specific central nervous system (CNS)
excitatory action, we tested the effects of gabazine on the
hypnotic effect of ketamine in 24 rats. Two animals were
excluded from the data analysis due to histological evi-
dence documenting that cannulae were placed outside the
right lateral ventricle.
Similar to isoflurane, IP ketamine produced LORR in
a dose-dependent fashion. However, in contrast to isoflu-
rane, ICV administration of gabazine did not affect the
LORR (neither the percentage nor the duration or onset
time) induced by any dose of ketamine (Figure 3). These

M. XIONG ET AL.
191
Figure 2. Systematic administration of isoflurane dose-
dependently induced LORR. (a) ICV administration of
gabazine (136 µM) produced a large reduction in the %
LORR induced by isoflurane, a significant rightward shift
of the dose response curve of isoflurane, and a significant
increase in the IC50 values of isoflurane (from 0.59% ±
0.04% to 0.78% ± 0.03%). Each point is the mean of the
results from tests on 10 - 14 rats; (b) Gabazine prolonged
the onset time of LORR. In those rats administered with
gabazine and evidence of LORR, significant prolongation of
onset time of LORR was observed at 0.6% and 0.9%
isoflurane. The values are expressed as mean ± S.E.M.
Number of rats in each group is indicated. *p < 0.05, ***p <
0.001 compared with saline groups (Paired t test).
results suggest that centrally administered gabazine se-
lectively blocks the effect of isoflurane, and the effect of
gabazine is not due to a non-specific CNS excitatory
action.
4. Discussion
Our study demonstrates that gabazine antagonized the
Figure 3. ICV administration of gabazine (136 µM) did not
affect the LORR induced by ketamine. Intraperitoneal in-
jection of ketamine dose-dependently induced LORR. (a)
ICV administration of gabazine (136 µM) did not affect the
percentage of LORR compared to ICV administration of
saline. (b) Onset time for LORR animals are not affected by
ICV gabazine injection. The values are expressed as mean ±
S.E.M. Number of rats in each group is indicated.
LORR induced by isoflurane, as evidenced by reducing
the percentage of rats exhibiting isoflurane-induced
LORR and by increasing the onset time in those animals
at 0.6% and 0.9% isoflurane.
In a previous study, Sugimura and colleagues reported
that bicuculline had no effect on the onset time of isoflu-
rane-induced LORR in mice [5]. While the mechanisms
underlying the discrepancy between the present study
and theirs required further study, there are several possi-
ble interpretations. In Sugimura’s study, they found that
picrotoxin can prolong the onset time of LORR induced
by isoflurane which is different from the effect of bicu-
culline. It is known that these two GABAAR antagonists

M. XIONG ET AL.
192
work via different mechanisms: picrotoxin is a non
competitive antagonist of GABAARs, while bicuculline
is a competitive antagonist of GABAARs. Therefore, the
different effect between these two agents observed in
mice may due to the difference between the competitive
and non-competitive nature. However, this interpretation
is not supported by the current study, since gabazine, si-
milar to bicuculline, is a competitive antagonist of
GABAARs [11]. The other possibility is that the differ-
ence between the methodologies in our study verses Su-
gimura’s study. In Sugimura’s study, only 2% of isoflu-
rane was tested. In our rat study, we tested a range of
lower concentrations (0.3% - 1.2%) of isoflurane. As
depicted in Figure 2, the antagonized effect of gabazine
tended to be stronger at the lower concentrations than
higher concentrations of isoflurane. Other possible con-
tributor to the discrepancy was the way the GABAAR
antagonists were administered. In Sugimura’s study, bi-
cuculline was administered by intraperitoneal injection,
while gabazine was injected into the cerebroventricular
space in the current study. In addition, bicuculline (free
base) is not soluble in aqueous solution, whereas the of-
ten-used N-methyl salts of bicuculline are not specific to
GABAA receptors [12]. Therefore, the efficacy and reli-
ability of delivery of IP administration of bicuculline to
brain GABAAR is challenging and difficult to predict. It
may require quite high dose of bicuculline IP injection in
order to observe any effect. However, high dose of bicu-
culline IP-injection may induce convulsion with ED50 of
4 mg/kg [the dose at which 50% of rats convulse] [13].
Therefore IP injection of bicuculline at high dosage se-
verely limited the feasibility to study the effect of bicu-
culline on LORR induced by isoflurane.
The current study has limitation. We could not clearly
identify whether LORR is due to loss mental status (se-
dation or hypnosis or unconsciousness) or immobilizing
action (spinal cord effect). Theoretically, ICV injection
of gabazine may also diffuse into spinal canal through
CSF. However, we believe that the LORR observed in
our study is largely due to the mental status change rather
than spinal effect for the following reasons. First, the
amount of gabazine in the present study is extremely low
(in the micromole range); therefore, the amount of gaba-
zine diffuse from cerebroventricle to spinal canal would
be very low to cause any detectable effect if there is any.
In other study, the IV infusion of gabazine (even at mi-
crogram range) has minimum effect on the suppressing
movement in response to a noxious stimulus under iso-
flurane [13]. In addition to that, the dose of general an-
esthetic required producing sedation and hypnosis is
much lower than that required to produce immobility.
For example, isoflurane prevents voluntary response to
spoken commands in human subject at concentrations that
are only 30 to 40% of the levels required for immobility
in response to a painful stimulus [14]. In our study, the
LORR occurred at fairly low concentration of isoflurane
[range of 0.6% - 1.2%] and immobility induced by
isoflurane is at a range of 1.5% - 2% [13]; therefore im-
mobility in our study may not be major contributor to the
LORR we observed. Second, our direct observation did
not support immobilizing action theory. We test the cor-
nea reflex in our rats that have LORR induced by isoflu-
rane. Rats have LORR also lost cornea reflex, indi-
cating it is more central brain effect rather than spinal
cord effect.
Further studies using GABAAR antagonists in specific
regions of the brain may help further delineate isoflu-
rane’s behavioral effects and mechanism of action. The
present study indicates that isoflurane is less effective as
a hypnotic in the presence of a GABAARs antagonist
(gabazine). We therefore conclude that the hypnotic ef-
fect of isoflurane is mediated, at least in part, by GAB-
AARs.
5. Acknowledgments
The authors appreciate Dr. Sheldon Goldstein for his
editing of the manuscript.
6. Financial Supports
This work is made possible by Foundation of University
of Medicine and Dentistry of New Jersey, Newark, NJ;
National Institute of Health, Bethesda, MD, grants:
AA015925, AT 001182 and AA016964. Parts of the con-
tents have been presented at meetings: Annual meeting,
American Society of Anesthesiology, October 13, 2007,
San Francisco, CA.
7. References
[1] U. Rudolph and B. Antkowiak, “Molecular and Neuronal
Substrates for General Anaesthetics,” Nature Reviews
Neuroscience, Vol. 5, No. 9, 2004, pp. 709-720.
Hdoi:10.1038/nrn1496
[2] L. E. Nelson, T. Z. Guo, J. Lu, C. B. Saper, N. P. Franks
and M. Maze, “The Sedative Component of Anesthesia Is
Mediated by GABA(A) Receptors in an Endogenous
Sleep Pathway,” Nature Neuroscience, Vol. 5, No. 10,
2002, pp. 979-984. Hdoi:10.1038/nn913
[3] G. Vanini, C. J. Watson, R. Lydic and H. A. Baghdoyan,
“Gamma-Aminobutyric Acid-Mediated Neurotransmis-
sion in the Pontine Reticular Formation Modulates Hyp-
nosis, Immobility, and Breathing during Isoflurane An-
esthesia,” Anesthesiology, Vol. 109, No. 6, 2008, pp. 978-
988. Hdoi:10.1097/ALN.0b013e31818e3b1b
[4] B. J. Saab, A. J. Maclean, M. Kanisek, A. A. Zurek, L. J.

M. XIONG ET AL.
193
Martin, J. C. Roder and B. A. Orser, “Short-Term Mem-
ory Impairment after Isoflurane in Mice Is Prevented by
the Alpha5 Gamma-Aminobutyric Acid Type A Receptor
Inverse Agonist L-655,708,” Anesthesiology, Vol. 113,
No. 5, pp. 1061-1071.
Hdoi:10.1097/ALN.0b013e3181f56228
[5] M. Sugimura, S. Kitayama, K. Morita, Y. Imai, M. Iri-
fune, T. Takarada, M. Kawahara and T. Dohi, “Effects of
GABAergic Agents on Anesthesia Induced by Halothane,
Isoflurane, and Thiamylal in Mice,” Pharmacology Bio-
chemistry and Behavior, Vol. 72, No. 1-2, 2002, pp. 111-
116. Hdoi:10.1016/S0091-3057(01)00728-6
[6] G. Paxinos and C. Watson, “The Rat Brain in Stereotaxic
Coordinates,” 6th Edition, Academic Press, Cambridge,
2007.
[7] D. R. Waud, “On Biological Assays Involving Quantal
Responses,” Journal of Pharmacology and Experimental
Therapeutics, Vol. 183, No. 3, 1972, pp. 577-607.
[8] N. P. Franks, “General Anaesthesia: From Molecular
Targets to Neuronal Pathways of Sleep and Arousal,”
Nature Reviews Neuroscience, Vol. 9, No. 5, 2008, pp.
370-386. Hdoi:10.1038/nrn2372
[9] N. P. Franks and W. R. Lieb, “Molecular and Cellular
Mechanisms of General Anaesthesia,” Nature, Vol. 367,
1994, pp. 607-614. Hdoi:10.1038/367607a0
[10] D. Lodge, N. A. Anis and N. R. Burton, “Effects of Op-
tical Isomers of Ketamine on Excitation of Cat and Rat
Spinal Neurones by Amino-Acids and Acetylcholine,”
Neuroscience Letters, Vol. 29, No. 3, 1982, pp. 281-286.
Hdoi:10.1016/0304-3940(82)90330-5
[11] S. Ueno, J. Bracamontes, C. Zorumski, D. S. Weiss and J.
H. Steinbach, “Bicuculline and Gabazine Are Allosteric
Inhibitors of Channel Opening of the GABAA Receptor,”
The Journal of Neuroscience, Vol. 17, No. 2, 1997, pp.
625-634.
[12] S. W. Johnson and V. Seutin, “Bicuculline Methiodide
Potentiates NMDA-Dependent Burst Firing in Rat Do-
pamine Neurons by Blocking Apamin-Sensitive Ca2+-
Activated K+ Currents,” Neuroscience Letters, Vol. 231,
No. 1, 1997, pp. 13-16.
Hdoi:10.1016/S0304-3940(97)00508-9
[13] J. M. Sonner, Y. Zhang, C. Stabernack, W. Abaigar, Y.
Xing and M. J. Laster, “GABA(A) Receptor Blockade
Antagonizes the Immobilizing Action of Propofol but Not
Ketamine or Isoflurane in a Dose-Related Manner,” An-
esthesia & Analgesia, Vol. 96, No. 3, 2003, pp. 706-712,
table of contents.
[14] R. Dwyer, H. L. Bennett, E. I. Eger and D. Heilbron,
“Effects of Isoflurane and Nitrous Oxide in Subanesthetic
Concentrations on Memory and Responsiveness in Vol-
unteers,” Anesthesiology, Vol. 77, No. 5, 1992, pp. 888-
898. Hdoi:10.1097/00000542-199211000-00009H
Abbreviations
Intracerebroventricular (ICV); Intraperitoneal (IP);
γ-aminobutyric acid A receptors (GABAARs);
loss-of-righting reflex (LORR)