Neuroscience 131 (2005) 79 – 86
ISOFLURANE INCREASES NOREPINEPHRINE RELEASE IN THE RAT
PREOPTIC AREA AND THE POSTERIOR HYPOTHALAMUS IN VIVO
AND IN VITRO: RELEVANCE TO THERMOREGULATION DURING
ANESTHESIA
T. KUSHIKATA,a* K. HIROTA,a N. KOTANI,b
H. YOSHIDA,a M. KUDOa AND A. MATSUKIa
a
Department of Anesthesiology, University of Hirosaki School of Med-
icine, Zaifu 5, Hirosaki, Japan
b
Department of Anesthesiology, Yamagata University Faculty of Med-
icine, Iida-Nishi 2-2-2, Yamagata, 990-955, Japan
Abstract—General anesthetics modulate autonomic nervous
system function including thermoregulatory control, which
resides in the preoptic area of the anterior hypothalamus.
However, the mechanism by which anesthetics modulate hy-
pothalamic function remains unknown. We hypothesized that
isoflurane increases norepinephrine release in the preoptic
area and in the posterior hypothalamus causing hypothermia
during anesthesia. To test this hypothesis, we performed a
series of in vivo and in vitro studies in rats. In vivo studies: 1)
Norepinephrine release was measured by microdialysis in
the preoptic area or the posterior hypothalamus (nⴝ9 each)
before, during (30 min), and after (50 min) rats were anesthe-
tized with 2% isoflurane. 2) In five rats, blood gases and
arterial pressure were measured. 3) Body temperature
changes (nⴝ6 each) were measured after prazosin (0, 0.05,
0.5 ␮g), norepinephrine (0, 0.1, 1.0 ␮g), or 0.5 ␮g prazosin
with 1.0 ␮g norepinephrine injection into the preoptic area. In
vitro study: Norepinephrine release was measured from an-
terior or posterior hypothalamic slices (nⴝ10 each) incubated
with 0, 1, 2, or 4% isoflurane in Ca2ⴙ-containing buffer or with
4% isoflurane (nⴝ10) in Ca2ⴙ-free buffer. Data were analyzed
with repeated measures or factorial ANOVA and Student–
Newman–Keuls tests. P<0.05 was significant. During anes-
thesia, norepinephrine release in the preoptic area was in-
creased approximately 270%, whereas the release in the poste-
rior hypothalamus remained unchanged. During emergence,
posterior hypothalamic norepinephrine release increased by
approximately 250% (P<0.05). Rectal temperature changes
correlated with norepinephrine release from the preoptic
area. Norepinephrine in the preoptic area enhanced isoflu-
rane-induced hypothermia, while prazosin reversed it. Nor-
epinephrine release from anterior hypothalamic slices in-
creased at all isoflurane concentrations, but only at the high-
est concentration in posterior hypothalamic slices. Under
Ca2ⴙ-free conditions, 4% isoflurane increased norepineph-
rine from both regions. These results suggest that augmen-
tation of norepinephrine release in the preoptic area is re-
sponsible for hypothermia during general anesthesia. © 2005
Published by Elsevier Ltd on behalf of IBRO.
*Corresponding author. Tel: ⫹81-172-39-5111; fax: ⫹81-172-39-5112.
E-mail address: masuika@cc.hirosaki-u.ac.jp (T. Kushikata).
Abbreviations: ANOVA, analysis of variance; AUC, area under the
curve; EDTA, ethylenediamine tetraacetic acid; EGTA, O,O=-bis(2-
aminoethyl)ethyleneglycol-N,N,N=,N=-tetraacetic acid; KRBS, Krebs–
Ringer bicarbonate buffer solution; PaO2, arterial oxygen pressure.
0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO.
doi:10.1016/j.neuroscience.2004.11.007
79
Key words: isoflurane, hypothalamus, norepinephrine, ad-
renergic receptor, microdialysis, thermoregulation.
All general anesthetics inhibit thermoregulatory control
(Sessler, 2000), which resides in the preoptic area of the
anterior hypothalamus; the mechanism by which they
moderate preoptic area function, however, remains un-
known.
Numerous mediators are involved in central thermo-
regulatory control; one of the most important of these is
norepinephrine. Studies in animals indicate that an in-
crease in preoptic hypothalamic norepinephrine provokes
hypothermia (Metcalf and Myers, 1978; Myers et al.,
1987). In the rat, for example, injection of norepinephrine
into the preoptic area of the anterior hypothalamus pro-
duces a dose-dependent decrease in core temperature
(Gisolfi and Christman, 1980). This hypothermia is re-
versed by ␣1-adrenergic antagonists such as phentol-
amine (Gisolfi and Christman, 1980).
We demonstrated previously that norepinephrine release
in the preoptic area of the anterior hypothalamus is increased
during isoflurane or sevoflurane anesthesia (Anzawa et al.,
2001) and during recovery from halothane (Chave et al.,
1996) or sevoflurane anesthesia (Ohkawa et al., 1995). How-
ever, the effect of anesthesia on the relationship between
thermoregulation and release of norepinephrine in the preop-
tic area of the anterior hypothalamus remains unknown. We
tested the hypothesis that isoflurane, a commonly used vol-
atile anesthetic, increases norepinephrine release in the pre-
optic area of the anterior hypothalamus and in the posterior
hypothalamus, and that this increase is responsible for hypo-
thermia during anesthesia. To test our hypothesis, in rats we
measured norepinephrine release in the preoptic area and in
the posterior hypothalamus both in vivo and in vitro upon
exposure to isoflurane. We additionally tested if the ␣1-ad-
renergic antagonist prazosin reverses isoflurane-induced hy-
pothermia.
EXPERIMENTAL PROCEDURES
The institutional committee on animal research at the University of
Hirosaki School of Medicine approved all protocols. Sixty-five
male Wistar rats (Japan Clea, Kyoto, Japan) weighing 280 –350 g
were used for the in vivo studies and 50 rats weighing 250 –300 g
were used in the in vitro study. All animals were housed for at least
1 week prior to the experiment and were kept on a 12-h light/dark
cycle (lights on 08:00 –20:00 h) at 22–24 °C with 40% humidity.
They had access to food and water ad libitum except for the day
of the experiment. All experiments were conducted between 11:00
80 T. Kushikata et al. / Neuroscience 131 (2005) 79 – 86

Table 1. Blood gas values and isoflurane concentration

Time (minutes after starting isoflurane)

0 10 20 30 40 50 60

pH 7.50⫾0.01 7.47⫾0.01* 7.48⫾0.01* 7.44⫾0.01* 7.47⫾0.01* 7.47⫾0.01* 7.48⫾0.01*

PaCO2 (mm Hg) 34.8⫾0.5 38.5⫾0.6* 37.3⫾0.3* 42.0⫾1.1* 36.5⫾0.9 35.4⫾1.0 36.6⫾0.7
PaO2 (mm Hg) 88.9⫾1.8 87.6⫾4.9 91.4⫾5.3 90.0⫾6.5 101.1⫾2.8* 95.3⫾2.8 96.7⫾2.5
Mean arterial pressure
(mm Hg) 107⫾3 91⫾4* 84⫾1* 90⫾2* 94⫾1* 96⫾1* 103⫾4
Blood isoflurane
concentration (mg/dl) ND 14.1⫾0.2 17.7⫾0.3† 18.2⫾0.2† 1.8⫾0.1† 0.9⫾0.1† 0.5⫾0.1†

* P⬍0.05 vs. Time 0; †P⬍0.05 vs. Time 10; ND, no data; PaCO2, arterial carbon dioxide pressure.

and 15:00 h since noradrenergic neuronal activity has a diurnal isoflurane inhalation for 50 min. An electrochemical detector with
rhythm. The procedures used were determined carefully to mini- a graphite electrode (ECD-300; EICOM) was employed for deter-
mize the number of animals used and their suffering. mining the amount of norepinephrine in the dialysates. Ten micro-
liters of each dialysate was used for high-performance liquid chro-
In vivo microdialysis study matography. The oxidation potential of the electrode was set at
⫹400 mV against an Ag/AgCl reference electrode. Norepineph-
Protocol. Eighteen rats were anesthetized with pentobarbi-
rine was separated on an ODS-C18 column (ø 2.1⫻150 mm,
tal (50 mg/kg intraperitoneally) and mounted in a stereotaxic frame
(David-Kopf brain fixation device, No. 30 –1000; BAS, Tokyo, CA-50 DS; EICOM) maintained at 25 °C. The mobile phase con-
Japan). A stainless-steel guide cannula (OD⫽0.5 mm, AG-12; sisted of 0.1 M phosphate buffer (pH 6.0) containing EDTA 200
EICOM, Kyoto, Japan) was implanted in the preoptic area of the mg/l, 1-octanesulphonate 400 mg/l, and 5% methanol. The flow
anterior hypothalamus (n⫽9) or the posterior hypothalamus rate of the mobile phase was 220 ␮l/min.
(n⫽9). The coordinates for the preoptic area were: AP: ⫺0.9, L:
0.5 mm in relation to the bregma, V: 8.5 mm from he skull surface. In vivo blood gas study
The guide cannula was implanted to the preoptic area from the
cortical surface at 11° from the vertical axis to prevent cortical Protocol. In five separate rats, we measured plasma isoflu-
surface vessel damage. Therefore, the coordinate of the guide rane concentration, arterial blood pressure, and blood gases.
cannula implantation were AP: ⫺0.9, L: 2.5 mm in relation to the Each animal was anesthetized with pentobarbital (50 mg/kg intra-
bregma. The coordinates for the posterior hypothalamus were: peritoneally) and an indwelling cannula inserted into the left fem-
AP: ⫺3.6 mm, L: 0.5 mm, V: 9.0 mm (Paxinos and Watson, 1986). oral artery. Twenty-four hours later, the animal was anesthetized
The cannula was fixed to the skull surface with dental cement with 2% isoflurane as described above. Blood samples for arterial
(Quick Resin; Shofu, Kyoto, Japan) and with two stainless-steel blood gas analysis and for measurement of plasma isoflurane
screws inserted above the cerebral cortex. Twenty-four hours concentrations were obtained 10 min before the start of isoflurane
after guide cannula implantation, a microdialysis probe anesthesia, every 10 min during the 30 min of isoflurane anesthe-
(OD⫽0.22 mm, A-I-12-2; EICOM) with a 2-mm semipermeable sia, and every 10 min for 30 min after cessation of isoflurane. The
membrane at its tip was inserted via the cannula. The tip of the removed blood volume was replaced with lactated Ringer’s solu-
probe protruded 2 mm above the tip of the guide cannula. The tion given through the femoral cannula.
probe tips were placed at the preoptic area or posterior hypothal-
amus, at the coordinates described above. After all experiments,
we verified the location of the probe histologically.
Twenty-four hours after probe insertion, rats were placed in
a custom built plastic box in which they could move freely (Free
Moving Unit CMA/125; BAS). To measure norepinephrine re-
lease in the preoptic area and the posterior hypothalamus, the
probe was perfused at a flow rate of 1.3 ␮l/min with artificial
cerebrospinal fluid of the following composition (in mM): NaCl
128, KCl 2.6, CaCl2 1.3, MgCl2 0.9, NaHCO3 20 Na2HPO4 1.3.
One millimolar of pargyline, a monoamine inhibitor, was in-
cluded to prevent degradation of norepinephrine. Dialysis sam-
ples were collected at 10-min intervals. After obtaining five
consecutive stable samples of norepinephrine, 2% isoflurane in
air was delivered into the box for 30 min at a flow rate of 4 l/min
to anesthetize the animal. A calibrated vaporizer (Forawick;
Muraco, Tokyo, Japan) was used and the concentrations of
isoflurane, oxygen, and carbon dioxide in the box were moni-
tored continuously with a gas analyzer (Capnomac; IMI, Tokyo,
Japan). After cessation of isoflurane inhalation, five additional
samples were obtained over 50 min. Fig. 1. Norepinephrine content in the dialysates from the preoptic
area (POA; filled circles; n⫽9) and posterior hypothalamus (PH; open
Measurements. Rectal temperature was measured with a triangles; n⫽9). The data are graphed as amount (pg) of norepineph-
thermorecorder (TR71S; T7D Corp., Nagano, Japan) 10 min be- rine per sample. Asterisk (*) indicates value is significantly different
fore the start of isoflurane anesthesia, every 10 min during the 30 (P⬍0.05) from control (average of all measurements from ⫺50 to 0
min of isoflurane anesthesia, and every 10 min after cessation of min).
 T. Kushikata et al. / Neuroscience 131 (2005) 79 – 86 81

Fig. 2. Changes in the rectal temperature with isoflurane anesthesia and linear regression between the temperature and changes in norepinephrine
release from the preoptic area-dialyzed group or posterior hypothalamus-dialyzed group. Isoflurane decreased rectal temperature during and for 40
min after it was given. The changes in the rectal temperature in the preoptic area-dialyzed group (POA; filled circles; n⫽9) and the posterior
hypothalamus-dialyzed group (PH; open triangles; n⫽9) were similar (A). Asterisk (*) indicates value is significantly different (P⬍0.05) from
pre-anesthesia control. The relationship between rectal temperature and norepinephrine release from the preoptic area was statistically significant
(solid line; B, P⬍0.05).

Measurements. The plasma isoflurane concentration was
measured by gas chromatography (G-5000A; Hitachi, Tokyo, Ja-
pan) using an equilibration method in a glass column (SE-30).
Temperature of column and detector were maintained at 105 °C
and 130 °C, respectively. The retention time of isoflurane was
60 s. The arterial blood pressure was measured with a polygraph
(PG 125; San-ei, Tokyo, Japan). The arterial blood gases and pH
were measured with a blood gas analyzer (ABL 510; Radiometer
Medical A/S, Copenhagen, Denmark).
In vivo microinjection study
Protocol. Forty-two rats were anesthetized and mounted in
a stereotaxic frame in the same manner as described above. The
82 T. Kushikata et al. / Neuroscience 131 (2005) 79 – 86
Fig. 3. Effect of microinjection of the ␣1 adrenergic antagonist prazo-
sin into the preoptic area on isoflurane-induced hypothermia. In A, time
course is shown. In B, AUC calculated from time ⫺10 through 80 min
is displayed. In A, the vehicle for prazosin, N-N-dimethyl acetamide,
did not affect rectal temperature when injected into the preoptic area
(open circles, 0 ␮g prazosin; n⫽6). In contrast, injection of 0.5 ␮g
prazosin (filled triangles; n⫽6) significantly increased rectal tempera-
ture. In B, after microinjection of 0.05 or 0.5 ␮g prazosin the AUC was
significantly increased (P⬍0.05).
same stainless-steel guide cannula as in the microdialysis study
was implanted in the preoptic area. The coordinates for the pre-
optic area were same as the microdialysis experiments described
above. The guide cannula was implanted at V 7.5 mm from the
skull surface. The tip of injector cannula protruded 0.5 mm above
the guide cannula so that the tip was placed above the preoptic
area. After a 1-week recovery period, one of following solutions
was injected into the preoptic area using a 30 gauge stainless
cannula (MI-12; EICOM): 0, 0.05, and 0.5 ␮g prazosin; 0, 0.1,
1.0 ␮g norepinephrine; or 0.5 ␮g prazosin with 1.0 ␮g norepineph-
rine. The groups designated as 0 ␮g prazosin or norepinephrine
were given the appropriate vehicle. Prazosin was dissolved in
10% N,N-dimethyl acetamide (Sigma Chemical, St. Louis, MO,
USA), and norepinephrine was dissolved in pyrogen-free physio-
logical saline. The injection volume was 0.4 ␮l and was done
manually over 4 min then the cannula was left in place for at least
1 min after the injection. Sixty minutes after the injection, rats were
anesthetized with 2% isoflurane as described above
Measurements. The rectal temperature was measured with
a thermorecorder (TR71S; T7D Corp.) before the injection, 10 min
before the start of isoflurane inhalation, every 10 min during 30
min of isoflurane anesthesia, and every 10 min after cessation of
isoflurane for 50 min.
In vitro study
Fifty conscious male Wistar rats (250 –300 g) were decapitated.
The brains were removed quickly and immersed in ice-cold Krebs–
Ringer buffer solution (KRBS). For Ca2⫹-containing conditions
(n⫽40), a KRBS with the following composition (in mM) was used:
NaCl 133, KCL 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.5, glucose
11.1, HEPES 10 (pH 7.4). For Ca2⫹-free conditions (n⫽10), a
KRBS with the following composition (in mM) was used: NaCl 133,
KCL 4.8, KH2PO4 1.2, MgSO4 1.2, glucose 11.1, HEPES 10 (pH
7.4), EGTA 1. The anterior hypothalamus, including the preoptic
area, or the posterior hypothalamus was dissected and chopped
with a tissue chopper to produce 350 ␮m slices. The slices were
washed three times with ice-cold KRBS and transferred to
polypropylene tubes (1 ml aliquots of slices were equivalent to
5– 6 mg protein). After discarding the supernatant, the slices were
resuspended in 1 ml of fresh KRBS and incubated for 10 min at
37 °C. This procedure was repeated. Immediately after the sec-
ond incubation, the slices were resuspended in 1 ml KRBS with
30% oxygen (control) and one of the following isoflurane concen-
trations: 0, 1, 2, or 4% for the Ca2⫹-containing conditions, 4%
isoflurane Ca2⫹-free conditions then incubated for additional 10
min at 37 °C.
Measurements
The norepinephrine content in each sample was determined by
high-performance liquid chromatography with an electrochemical
detector (Coulochem Model 5100 A; ESA, Tokyo, Japan). Twenty
microliter aliquots of the samples, acidified with perchloric acid,
were injected onto a reverse-phase column (C18, 4.6⫻150 mm;
MC Medical, Tokyo, Japan). Norepinephrine was separated by
use of a mobile phase buffer consisting of 10% acetonitrile, 5%
methanol, and 85% 0.05 M NaH2PO4, 0.05 M CCl3COOH (trichlo-
roacetate), 0.7 mM CH3(CH2)11OSO3Na (sodium dodecyl sulfate)
and 0.02 mM EDTANa2 (EDTA disodium) at pH 3.4 and a flow rate
of 1 ml/l at 40 °C. Norepinephrine was quantified using an elec-
trochemical detector set at 300 mV. Under these conditions, the
retention time for measurement of norepinephrine content was 6.3
min.
Statistical analyses
Induction time was defined as the time from the start of isoflurane
inhalation to loss of the righting reflex. Recovery time was defined
as the time from cessation of isoflurane inhalation to regaining of
the reflex (Dickinson et al., 2000). Data from the microdialysis
studies, except for induction and recovery times, were analyzed
by repeated measures ANOVA followed by Student–Newman–
Keuls tests. Induction and recovery times were analyzed by Stu-
dent’s unpaired t-tests. Regression analysis was used to deter-
mine the relationship between changes in the rectal temperature
and maximum values of norepinephrine release from the preoptic
area or the posterior hypothalamus during isoflurane inhalation.
The changes in rectal temperature were calculated as the maxi-
mum or minimum temperature during isoflurane inhalation minus
the pre-inhalation value. In the microinjection study, the area
under the curve (AUC) of the rectal temperature time course from
pre-injection to 50 min after isoflurane inhalation was measured
with computer-based software as in our previous study (Yoshida
et al., 2001) and analyzed by repeated measures ANOVA fol-
lowed by Student–Newman–Keuls tests. Norepinephrine values
obtained in the in vitro study were converted to the percent of each
animal’s control value and analyzed by factorial ANOVA followed
by Student–Newman–Keuls tests. All data are expressed as
means⫾S.E.M.; P⬍0.05 was considered statistically significant.
 T. Kushikata et al. / Neuroscience 131 (2005) 79 – 86 83

Fig. 4. Effect of norepinephrine microinjection into the preoptic area on isoflurane-induced hypothermia. In A, time course is shown. In B, AUC
calculated from time ⫺10 through 80 min is displayed. Rectal temperature decreased following isoflurane inhalation in the control (open circles, 0 ␮g
norepinephrine; n⫽6). In B, the AUC after a 1.0 ␮g-norepinephrine microinjection was significantly decreased and the decrease was reversed by
co-administered 0.5 ␮g prazosin. Asterisk (*) indicates significant difference from control (P⬍0.05).

RESULTS rane administration compared with 10 min. The concentra-

The mean induction time was 5.2⫾0.4 min in the preoptic
area-dialyzed group and 6.0⫾0.4 min in the posterior hy-
pothalamus-dialyzed group. The recovery time was
7.5⫾0.7 min in the preoptic area group and 6.8⫾0.4 min in
the posterior hypothalamus group. There were no signifi-
cant differences between the groups for either parameter.
Arterial pH decreased during the first 10 min of the
isoflurane anesthesia and remained low until 30 min after
cessation (P⬍0.05), whereas arterial carbon dioxide pres-
sure increased only during the anesthesia period
(P⬍0.05). In contrast, there were no significant decreases
in arterial oxygen pressure (PaO2) at any time. Mean
arterial blood pressure declined during isoflurane anesthe-
sia and remained low through the first 20 min of emer-
gence (P⬍0.05). The arterial isoflurane concentration was
significantly greater at 20 and 30 min after start of isoflu-
tion decreased to less than the 10-min concentration im-
mediately after cessation of isoflurane inhalation (Table 1).
Norepinephrine content in the dialysates from the pre-
optic area was stable in the five samples from all animals
before giving isoflurane (⫺50 – 0 min on Fig. 1). These
values were thus averaged to obtain the control norepi-
nephrine level of 1.04⫾0.10 pg. However, after the first 10
min of isoflurane anesthesia, the norepinephrine level in-
creased markedly to 2.72⫾0.49 pg (P⬍0.05) and re-
mained elevated through the recovery period (P⬍0.05
compared with the control value). Norepinephrine content
of the dialysates from the posterior hypothalamus was also
stable in the five samples from all animals before giving
isoflurane; these were averaged therefore to give a control
value of 0.82⫾0.08 pg. Norepinephrine content increased
slightly during isoflurane anesthesia, but did not differ sta-
84 T. Kushikata et al. / Neuroscience 131 (2005) 79 – 86

Fig. 5. A typical probe location in the preoptic area (A) and the posterior hypothalamus (B). Arrow indicates the location of the probe tip in each
region.

tistically from control. In contrast, norepinephrine content vehicle. The decrease was reversed by co-administered
in the sample taken 10 min after cessation of isoflurane 0.5 ␮g prazosin (Fig. 4B).
anesthesia (2.00⫾0.48 pg) exceeded the control value A typical figure indicating probe placement in the pre-
(P⬍0.05; Fig. 1). optic area or posterior hypothalamus is shown in Fig. 5
The changes in the rectal temperature in the preoptic Under Ca2⫹-containing conditions, norepinephrine re-
area-dialyzed group or in the posterior hypothalamus- lease from anterior hypothalamus slices increased signifi-
dialyzed group were similar. The average rectal temper- cantly during exposure to 1%, 2%, and 4% isoflurane.
ature of the groups before isoflurane anesthesia was
Norepinephrine contents from the posterior hypothalamus
38.0⫾0.1 °C. The temperature decreased significantly
slices only differed significantly from control during expo-
during 2% isoflurane inhalation and started to increase
sure to 4% isoflurane (Fig. 6).
10 min after its cessation. The temperature remained
significantly lower than control until 30 min after emer- Under Ca2⫹-free conditions, 4% isoflurane significantly
gence from the anesthesia (Fig. 2A). Regression anal- increased norepinephrine release from the anterior hypo-
ysis showed a significant relationship between the thalamus slices compared with 0% (227⫾24% vs.
changes in rectal temperature and norepinephrine re- 107⫾6%, P⬍0.01). Norepinephrine content increased sig-
lease from the preoptic area (straight line; P⬍0.05; nificantly in posterior hypothalamus slices as well
r2⫽0.55) but not from the posterior hypothalamus (119⫾3% vs. 106⫾5%, P⬍0.05).
(hatched line; Fig. 2B).
In the microinjection study, control rectal temperatures
were similar among all experimental groups. The injection
of N,N-dimethyl acetamide, the vehicle for prazosin, into
the preoptic area did not affect body temperature before
isoflurane administration. The maximum changes in the
rectal temperature during isoflurane were ⫺1.0⫾0.1 °C
with N,N-dimethyl acetamide, ⫺0.5⫾0.1 °C with 0.05 ␮g
prazosin, and 0.2⫾0.2 °C with 0.5 ␮g prazosin. Prazosin
alone at the highest dose (0.5 ␮g) increased rectal tem-
perature by 0.6⫾0.2 °C before isoflurane administration
(Fig. 3A). The AUC of the rectal temperature time course
from ⫺10 to 80 min was significantly increased after mi-
croinjection of 0.05 or 0.5 ␮g prazosin (Fig. 3B).
Injection of pyrogen-free physiological saline, the
vehicle for norepinephrine, into the preoptic area did not
affect body temperature. The maximum changes in the
rectal temperature during isoflurane were ⫺1.0⫾0.2 °C
with the vehicle, ⫺0.9⫾0.2 °C with 0.1 ␮g norepineph-
rine, ⫺1.95⫾0.2 °C with 1.0 ␮g norepinephrine. The
highest dose of norepinephrine alone (1.0 ␮g) de- Fig. 6. Norepinephrine release from brain slices in Ca⫹2-containing
creased rectal temperature by ⫺0.5⫾0.2 °C before conditions in the presence of 0, 1, 2, and 4% isoflurane. Norepi-
isoflurane administration. Co-administered prazosin an- nephrine is expressed as percent of control release. Solid boxes
tagonized the norepinephrine-induced hypothermia (Fig. indicate norepinephrine released from anterior hypothalamic slices
(n⫽10 each), including the preoptic area; open boxes indicate
4A). The AUC of the rectal temperature time course from norepinephrine release from posterior hypothalamic slices (n⫽10
⫺10 to 80 min after norepinephrine 1.0 ␮g microinjec- each). Asterisk indicates significant difference from control (0%
tion was significantly decreased compared with that with isoflurane; * P⬍0.05).
 T. Kushikata et al. / Neuroscience 131 (2005) 79 – 86
DISCUSSION
Noradrenergic mechanisms in the preoptic area of the
anterior hypothalamus play a significant role in thermoreg-
ulatory control of most animals (Cooper et al., 1976; Poole
and Stephenson, 1979; Metcalf and Myers, 1978; Gisolfi
and Christman, 1980; Myers et al., 1987; Mallick and
Joseph, 1998; Ramesh and Kumar, 1998; Mallick et al.,
2002); noradrenergic neurons are also involved in auto-
nomic nervous system control of cardiovascular function
both in the preoptic area of the anterior hypothalamus and
in the posterior hypothalamus (Sawchenko and Swanson,
1981; Guyenet, 1991; Osborne and Kurosawa, 1994;
Bealer, 1999; Kaehler et al., 1999). Our study evaluated
the release of norepinephrine in these two brain areas
before, during, and after isoflurane administration.
We found that in vivo isoflurane anesthesia increased
the amount of norepinephrine in dialysates collected from
the preoptic area of rats. In contrast, the amount of nor-
epinephrine in dialysates from the posterior hypothalamus
increased significantly only after the cessation of isoflu-
rane. The discrete sensitivity to isoflurane may reflect the
different physiologic role of each region.
Local application of norepinephrine to the preoptic area
induces hypothermia in rats (Poole and Stephenson,
1979), and when prazosin is injected into the preoptic area
in rats, rectal temperature increases (Mallick and Alam,
1992). Also, prazosin significantly inhibits the firing rate of
thermosensitive neurons (Mallick et al., 2002). These re-
sults indicate that noradrenergic neurons in the preoptic
area are involved in thermoregulation via the ␣1-adreno-
ceptor. Therefore, our observed changes in norepineph-
rine release in the preoptic area might contribute to isoflu-
rane-induced hypothermia. Indeed, we found that prazosin
injection into the preoptic area prevented isoflurane-in-
duced hypothermia whereas norepinephrine injection
evoked hypothermia. This suggests that increases in nor-
epinephrine release in the preoptic area may be responsi-
ble, at least in part, for isoflurane-induced hypothermia via
␣1-adrenoceptor.
The increased norepinephrine release in the posterior
hypothalamus during emergence from isoflurane anesthe-
sia is consistent with our previous finding that norepineph-
rine release increases markedly in the posterior hypothal-
amus during emergence from halothane or sevoflurane
anesthesia (Ohkawa et al., 1995; Chave et al., 1996).
Because noradrenergic neuronal activity in the posterior
hypothalamus is responsible for cardiovascular regulation
(Bealer, 1999; Kaehler et al., 1999), the increase in nor-
epinephrine release observed in the posterior hypothala-
mus may be related to sympathetically mediated actions
such as the increases in arterial blood pressure and heart
rate seen during emergence from isoflurane anesthesia. In
addition, the posterior hypothalamus plays a key role in the
control of body temperature through the sympathetic ner-
vous system. For example, core temperature was in-
creased by the local application to the posterior hypothal-
amus of the excitatory amino acid glutamate (Amir, 1990).
The activity of the posterior hypothalamus neurons is re-
85
lated to sympathetic nerve tone and body temperature
induced by PGE1 i.c.v. injection (Monda et al., 1994). As
Prostaglandin E i.c.v. injection affects posterior hypotha-
lamic norepinephrine accompanied by changes in body
temperature and firing rate of the posterior hypothalamus
in rats (Monda et al., 2000), the norepinephrine could be
involved in thermoregulation such as thermogenesis.
Therefore, our result of increases in norepinephrine re-
lease during emergence from isoflurane anesthesia sug-
gested that the posterior hypothalamic norepinephrine
could also be involved thermoregulation during emergence
from isoflurane anesthesia.
Our in vitro study showed that in the presence of 4%
isoflurane norepinephrine release from both anterior and
posterior hypothalamic brain slices increased. At lower
isoflurane concentrations, the increased release was seen
only in anterior hypothalamic slices. In Ca2⫹-free buffer,
only the highest concentration of isoflurane caused a sig-
nificant increase in norepinephrine release in either region.
This suggests that not only the extracellular calcium but
also the intracellular calcium store was responsible for the
release of the norepinephrine. This is consistent with a
previous study showing that halothane or isoflurane in-
creases the intracellular concentration of Ca2⫹ in the rat
cerebral cortex and hippocampus. This occurs predomi-
nantly by inducing release of Ca2⫹ from intracellular sites
rather than a Ca2⫹ influx from the extracellular medium
(Kindler et al., 1999). An increase in the intracellular Ca2⫹
concentration affects many neurotransmission and intra-
cellular signaling pathways, such as ion channel activity or
second-messenger systems, and thus it is an important
component of the mechanism of anesthesia.
Hypoxia affects norepinephrine metabolism in the CNS
(Roubein et al., 1981). However, in our study, there was
not a significant decrease in PaO2 during isoflurane anes-
thesia in vivo, nor was low oxygen likely to develop in vitro
since the KRBS was oxygenated with 30% oxygen. Hypo-
tension also affects norepinephrine metabolism. Hypoten-
sion (MAP⬍75 mm Hg) increases norepinephrine release
in the rat paraventricular/anterior hypothalamic region
(Van Huysse and Bealer, 1991). In our study, such hypo-
tension was not observed during isoflurane anesthesia or
emergence. In addition, according to the changes in the
blood isoflurane concentration, the induction time, and the
recovery time, the rats reached the anesthetic plane during
first 10 min of isoflurane anesthesia and recovered within
10 min of stopping anesthesia. Therefore, the changes in
norepinephrine concentration observed in both in vivo and
in vitro studies appeared to be caused by exposure to
isoflurane anesthesia itself.
We conclude that isoflurane anesthesia causes distinct
effects on norepinephrine release in the rat preoptic area
and posterior hypothalamus. The augmentation of norepi-
nephrine release in the preoptic area may be responsible
for the well-known hypothermia that occurs during general
anesthesia. In contrast, the increase in norepinephrine
release during emergence from isoflurane may be at least
partially responsible for the cardiovascular effects and
86 T. Kushikata et al. / Neuroscience 131 (2005) 79 – 86
thermogenesis associated with recovery from general
anesthesia.
Acknowledgments—We thank Dr. Daniel I. Sessler for his scien-
tific advice on this research. This study was supported, in part, by
the Ministry of Education, Science and Culture, Tokyo, Japan
(Grant-in-aid numbers 09470323 and 15659366).
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