Neuroscience 223 (2012) 277–284

INHIBITORY EFFECT OF ACTIVATION OF GABAA RECEPTORIN THE CENTRAL NUCLEUS OF AMYGDALA ON THE SODIUM INTAKEIN THE SODIUM-DEPLETED RAT

Q. WANG, a,b J. R. LI, a X. J. YANG, a K. CHEN, a

B. SUN a AND J. Q. YAN a*

aDepartment of Physiology and Pathophysiology, Key Laboratory

of Environment and Genes Related to Diseases, Ministry of

Education, Xi’an Jiaotong Universit College of Medicine,

76# W. Yanta Road, Xi’an, Shaanxi 710061, PR China

bDepartment of Physiology, Xi’an Medical University,

1# Xinwang Road, Xi’an, Shaanxi 710021, PR China

Abstract—The present study investigated the effects of a

microinjection of GABAA receptor agonist (muscimol) and

antagonist (bicuculline) into the central nucleus of the

amygdala (CeA) in sodium-depleted rats. We measured the

sodium intake and identified the neuronal activation in the

brainstem induced by activating the GABAA receptors in

the CeA using Fos immunohistochemistry. Muscimol (0.20,

0.35 or 0.50 nmol, in 0.2 ll) that was injected bilaterally into

the CeA decreased the 0.3 M NaCl and water intake in a

dose-dependent manner. Microinjection of 0.02 nmol/0.2 llmuscimol also decreased the NaCl intake, but had no effect

on the water intake. The inhibitory effect of muscimol

(0.20 nmol) on the sodium and water intake could be

blocked by pretreatment with bicuculline intra-CeA microin-

jection (0.4 nmol). However, bilateral injections of bicucul-

line alone into the CeA did not affect the NaCl or water

intake. Furthermore, microinjection of muscimol (0.20 nmol)

into the CeA increased the number of Fos-like immunoreac-

tive (FLI) neurons in the caudal and intermediate parts of the

nucleus of the tractus solitarius (cNTS and iNTS) and the lat-

eral parabrachial nucleus (LPBN). These results suggest

that GABAA receptors within the CeA may be involved in

mediating the sodium intake in the sodium-depleted rat,

and the cNTS, iNTS and LPBN were probably involved in this

mechanism. � 2012 IBRO. Published by Elsevier Ltd. All

rights reserved.

Key words: sodium appetite, GABAA receptors, parabrachial

nucleus, amygdala, nucleus tractus solitarius, Fos immuno-

histochemistry.

0306-4522/12 $36.00 � 2012 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2012.07.068

*Corresponding author. Tel/fax: +86-2982655199.

E-mail address: jqyan810@gmail.com (J. Q. Yan).Abbreviations: AP, area postrema; CeA, central nucleus of amygdala;cNTS, the caudal NTS; dl-PBN, dorsal lateral subnucleus of PBN;el-PBN, external lateral subnucleus of PBN; FLI, Fos-likeimmunoreactive; GABA, gamma-aminobutyric acid; iNTS, intermediateNTS; LPBN, lateral parabrachial nucleus; MPBN, medial parabrachialnucleus; mNTS, medial NTS; NTS, nucleus tractus solitaril; PBN,parabrachial nucleus; rNTS, rostral NTS.

277

INTRODUCTION

Sodium ions are positively charged ions in extracellular

fluid that are a major component in the maintenance of

body fluid homeostasis. Under sodium deprivation

conditions, animals display an increase in the salt intake

over a wide range of sodium chloride concentrations

(Weisinger et al., 1982; Berridge et al., 1984; Prakash

and Norgren, 1991). The central nucleus of the amygdala

(CeA) in the forebrain is the most important center for the

integration of gustatory and visceral signals (McDonald,

1998) and a lesion to CeA has been proved to influence

the sodium appetite of animals (Galaverna et al., 1992,

1993; Seeley et al., 1993; Zardetto-Smith et al., 1994). A

line of evidence has indicated that gamma-aminobutyric

acid (GABA) modulates the sodium and water intake via

GABAA receptors in some brain regions (Jones and

Mogenson, 1982; Unger et al., 1983; Abe et al., 1988;

Tanaka et al., 2003), and that microinjection of GABAA

receptor agonists or antagonists into the CeA can

produce the opposite effects on food intake (Minano

et al., 1992). A previous report from our laboratory

suggested that the GABAA receptors within the CeA

participate in modulating the responsiveness of gustatory

neurons in the parabrachial nucleus (PBN) (Kang et al.,

2004). It has also been reported that GABAA receptors

within the lateral parabrachial nucleus (LPBN) are

involved in the regulation of NaCl intake (Callera et al.,

2005; de Oliveira et al., 2007; Kimura et al., 2008). These

findings suggest that the CeA may modulate sodium

appetite at least partly via GABAA receptors. The CeA

contains large populations of GABAergic neurons and

terminals (Nitecka and Ben-Ari, 1987; Araki et al., 1992;

McDonald and Augustine, 1993; Sun and Cassell, 1993).

However, it is unclear whether the GABA receptors within

the CeA regulate the sodium appetite in the sodium-

depleted rat.

Previous studies have shown that the nucleus tractus

solitaril (NTS) and PBN not only are involved in the

excitatory regulation of sodium intake, but also in the

inhibitory control of sodium and water intake induced by

sodium depletion (Menani et al., 1998; Andrade et al.,

2004; Geerling et al., 2006; David et al., 2008).

Moreover, the anatomical and electrophysiological

evidences have shown that reciprocal connections exist

among the CeA, PBN and NTS (Saha et al., 2000;

Geerling and Loewy, 2006), and that the descending

projections from the CeA to the brainstem play an

important role in the regulation of taste (Li et al., 2002,

d.

278 Q. Wang et al. / Neuroscience 223 (2012) 277–284

2005; Cho et al., 2003; Huang et al., 2003). Vendramini’s

findings explored that damage to the CeA produced a

marked increase in c-Fos expression in the medial

parabrachial nucleus (MPBN) in the water deprivation-

partial rehydration of rats (Vendramini et al., 2009).

However, it is unknown whether the modulation of the

brainstem by the CeA is involved in the regulation of

sodium intake induced by sodium depletion.

To address the above issues, the present study

examined the effects of intra-CeA microinjection of

GABAA receptor agonist or antagonist on the sodium

and water intake behavior, as well as on the Fos-like

immunoreactive (FLI) expression in the related nuclei of

the brainstem in the sodium-depleted rat induced by

furosemide.

EXPERIMENTAL PROCEDURES

Animal surgery

Adult male Sprague–Dawley rats (provided by Medical

Experimental Animal Center of Xi’an Jiaotong University,

Shaanxi Province, China) weighing between 230 and 270 g

were housed individually in stainless-steel cages with free

access to food pellets and distilled water. The rats were

exposed to 12 h of light per day and the room temperature was

maintained at 25 ± 1 �C. Baseline sodium (0.3 M NaCl

solution) intake was measured 3 days before surgery. Rats with

a baseline sodium intake of over 15 g per day were excluded

from further experiment.

After anesthesia with chloral hydrate (300 mg/kg,

intraperitoneally, i.p.), each rat was secured in a stereotaxic

apparatus. Two stainless steel 23-gauge cannulas were

bilaterally implanted into the brain with their tips positioned at

the points 1 mm above the CeA (2.25–2.50 mm caudal to the

bregma, 4.0–4.2 mm lateral to the midline, 6.70–7.20 mm below

the dura mater) (Paxinos and Watson, 2005). The cannulas

were fixed to the skull by four screws and dental acrylic resin.

A 30-gauge metal obturator filled the cannulas between tests.

All rats were injected with penicillin (20,000 units, i.p.) during

the first 3 postoperative days to prevent infection and were

allowed to recover for at least 7 days before the beginning of

ingestion tests. The experimental protocol was in accordance

with the Principles of Laboratory Animal Care (NIH Publication

No. 85-23) and was approved by the Institutional Animal Care

Committee of Xi’an Jiaotong University. All efforts were made

to minimize the number of animals used and animal distress.

Drug microinjection

Muscimol HBr (a GABAA receptor agonist, Sigma–Aldrich, St.

Louis, MO, USA) and bicuculline (a GABAA receptor

antagonist, Sigma–Aldrich) were dissolved in 0.9% sodium

chloride immediately before the experiments. The drugs were

microinjected bilaterally into the CeA at an interval of 1 min

using 1 ll microsyringes which extended 1.0 mm beyond the

tips of the guide cannulas. Each injection lasted for 30 s and

the syringe was left in place for an additional 1 min. After the

injections, the obturators were replaced and the rats were

placed back into their cages.

Behavioral tests

Post-surgery rats underwent 4-day training for the two-bottle

choice test between water and 0.3 M NaCl. The rats drank from

the two bottles for 3 h in the morning (8:00–11:00) and for 1 h

in the afternoon (17:00–18:00) every day. For the rest of the

day, the rats were deprived of any drinking water. The water

and sodium solution intakes were recorded daily. On the 5th

day, all rats were sodium depleted by subcutaneous (s.c.)

injection of furosemide (20 mg/kg,) at 8:00 and fed with sodium

deficient food for 24 h.

On the 6th day, one part of the sodium-depleted rats were

randomly divided into five groups (n= 8/group) and 0.02, 0.2,

0.35, and 0.5 nmol muscimol and saline (0.2 ll) were

microinjected bilaterally into the CeA, respectively. The rats

underwent a two-bottle choice test (water verse 0.3 M NaCl

solution) and the cumulative intakes of water and 0.3 M NaCl

solution were recorded at 15, 30, 60, 120 and 180 min after the

microinjections. The concentration of muscimol which had a

moderate effect on the sodium intake in rats was used in the

following experiment.

Another part of the sodium-depleted rats were randomly

divided into four groups (n= 8/group). The four groups of

rats received two bilateral CeA injections (0.2 ll/injection) of

normal saline plus normal saline (NS + NS), normal saline

plus muscimol (0.2 nmol) (NS +mus), bicuculline (0.4 nmol)

plus normal saline (bic + NS), and bicuculline (0.4 nmol) plus

muscimol (0.2 nmol) (bic + mus), respectively, at an interval of

15 min. Then, the rats underwent a two-bottle choice test. The

cumulative intakes of water and 0.3 M NaCl solution were

recorded at 15, 30, 60, 120 and 180 min after the microinjections.

Immunohistochemical test

Immunohistochemistry of c-Fos expressions in the brains of

sodium-depleted rats after bilateral CeA injections of muscimol

was performed to localize the brain regions involved in the

sodium intake.

Fourteen post-operative rats were treated with furosemide

(20 mg/kg s.c.) and then fed with sodium-deficient food and

water. Twenty-four hours later, the rats received a bilateral CeA

injection of 0.2 ll muscimol (0.2 nmol/0.2 ll, n= 8) or saline

(n= 6). After being left undisturbed for 90 min, all rats were

anesthetized with an overdose of urethane and perfused

transcardially with 100 ml of 0.01 M phosphate-buffered saline

(PBS, pH 7.4), followed by 400 ml 4% paraformaldehyde in

0.1 M phosphate buffer (PB, pH 7.4). The brains were removed

immediately and post-fixed in the same fresh fixative for 4 h

before being soaked overnight at 4 �C in a 30% sucrose

solution in 0.1 M PB (pH 7.4). Subsequently, coronal brain

sections were cut at a thickness of 40 lm using a freezing

microtome (Leica, Germany), collected in 0.01 M PBS and

placed into two different dishes according to their numerical

orders of cutting (e.g. sections 1, 3, 5 to one dish; sections 2,

4, 6 to the other dish).

The streptavidin-peroxidase-conjugated method was used

for Fos immunohistochemistry. All immunohistochemical

procedures were performed at room temperature unless

otherwise noted, and 0.01 M PBS (pH 7.4) was used for all

rinses. The coronal sections in the first dish were washed

carefully and then incubated in 0.3% H2O2 for 20 min, rinsed

three times (5 min/rinse), incubated in 0.3% Triton X-100

diluted in 0.01 M PBS (pH 7.4) for 20 min and rinsed three

times again. Afterward, the sections were incubated in 10%

normal goat serum for 1 h prior to primary antibody incubation

with a rabbit polyclonal Fos antiserum (Abcam, AB7963-1,

Cambridge, UK) diluted 1:800 in 0.01 M PBS (pH 7.4) for 72 h

at 4 �C. After being rinsed three times (10 min/rinse), a rabbit

streptavidin-peroxidase staining kit (Zhongshan Bio-tech Co.,

Ltd., Beijing, China) was used for biotinylated secondary

antibody and horseradish peroxidase–avidin (egg white)

conjugate incubation according to the manufacturer’s

instructions. The Fos protein immunoreactive products were

visualized by using 3,30-diaminobenzidine tetrahydrochloride

(Zhongshan Bio-tech Co., Ltd., Beijing, China) as chromogen.

Q. Wang et al. / Neuroscience 223 (2012) 277–284 279

After that, the sections were mounted onto poly-L-lysine-coated

glass slides, air dried, dehydrated, coverslipped, and observed

under a light microscope (Olympus BX-51, Japan). Images

were acquired using a digital camera attached to the

microscope. The number of FLI neurons in the brainstem

regions of NTS and PBN was bilaterally counted by an

experimenter blind to the purpose of the experiment. For each

rat, approximately six coronal sections of each region were

analyzed and the immunohistochemical results were averaged.

FLI neurons in the NTS and the PBN were identified using the

light microscope. The sections in the second dish incubated in

PBS instead of the primary antibody or biotinylated goat anti-

rabbit IgG were used as negative controls, which did not exhibit

any of the staining described in this report.

Histology

At the end of the experiments, 2% Pontamine Sky Blue solution

(0.2 ll) was infused into each rat in the same way as the drugs

were injected. The rats were then given an overdose of urethan

and perfused transcardially with PBS, followed by 10% buffered

formalin. The brains were removed, fixed, and frozen-sectioned

(40 lm) in a coronal plane, and stained with Cresyl Violet. The

sites of the drug injections were identified according to Paxinos

and Watson (2005).

Statistical analysis

All data are indicated as means ± SEM. Two-way repeated-

measures of analysis of variance (two-way RM ANOVA)

followed by a post hoc multiple comparison was used to

analyze the cumulative water and 0.3 M NaCl intake in different

groups and times. Linear regression was performed to analyze

the correlation between drug dose and effect and count the

ED50. The independent samples t-test was used for statistical

cross-group comparison of the number of FLI neurons after

intra-CeA microinjection of muscimol or saline. All statistical

analyses were performed using Statistical Program for Social

Sciences statistical software (SPSS 16.0). The level of

statistical significance was set at P< 0.05.

RESULTS

Histological analysis

Fig. 1 shows the correct cannula placement in the CeA,

corresponding to �2.25 to �2.50 mm from bregma

according to the placement coordinates described in the

Paxinos and Watson (2005). Most of the injections were

localized in the lateral and medial portions of the CeA. A

total of 141 rats were used in these experiments, and

the histological analyses showed that 86 of them had

bilateral injections correctly made into the CeA. The

data from the 86 rats were used for the following

analyses.

Since there was no difference in water or sodium

intake between two-sided misplaced injection of the

muscimol and saline group, the data from the animals in

which the injection sites were not correctly placed within

the CeA were not analyzed.

Inhibitory effects of bilateral CeA injections ofmuscimol on 0.3 M NaCl and water intakes in sodium-depleted rats

Under saline control conditions, the sodium-depleted rats

induced by furosemide approached the 0.3 M NaCl and

water bottles almost immediately after the bottles were

placed on the cages and consumed most of their total

liquids during the first 45-min measurement period.

Sixty-seven percent (67%, 22.7/33.8 ml) of the total

liquid intake was from the 0.3 M NaCl bottle, which was

significantly larger than that from the water bottle

(P< 0.001). However, bilateral microinjections of

muscimol (0.02, 0.20, 0.35 or 0.5 0 nmol, in 0.2 ll) intothe CeA remarkably decreased 0.3 M NaCl and water

intake in the sodium-depleted rats in a dose-dependent

manner (r= 0.982, P= 0.003, ED50 = 0.23 nmol for

0.3 M NaCl intake; r= 0.997, P< 0.001, ED50 =

0.26 nmol for water intake) during the 180-min

observation period, As shown in Fig. 2A, B, the time

course curves (i.e., saline control and different doses of

muscimol-treated groups) were significantly different

between treatments (F(4,175) = 69.73, P< 0.001 for

0.3 M NaCl intake; F(4,173) = 28.05, P< 0.001 for water

intake), across time (F(5,175) = 118.89, P< 0.001 for

0.3 M NaCl intake; F(5,175) = 123.89, P< 0.001 for

water intake) and time � treatment (F(20,175) = 13.13,

P< 0.001 for 0.3 M NaCl intake; F(20,175) = 12.09,

P< 0.001 for water intake). Further analyses indicated

that the 0.3 M NaCl intake in different doses of

muscimol-treated groups were significantly smaller than

those in the saline control group (P< 0.05) at every

time point (Fig. 2A), but the water intake in smallest

dose (0.02 nmol) of the muscimol-treated group was not

significantly different from that of the saline control

group at every time point (Fig. 2B). Further observation

found that the 0.3 M NaCl consumption had no

significant difference on the muscimol-treated group and

the saline control group at 24 h (P> 0.05, data not

shown).

Blocking effects of bicuculline on muscimol-inducedinhibition of the 0.3 M NaCl and water intakes insodium-depleted rats

Bicuculline (0.4 nmol, in 0.2 ll) microinjected into the CeA

15 min prior to muscimol injection completely blocked the

muscimol (0.2 nmol, in 0.2 ll)-evoked inhibition of the

0.3 M NaCl and water intakes. However, microinjection

of bicuculline alone into the CeA did not affect the 0.3 M

NaCl or water intakes. Statistical analyses indicated that

the 0.3 M NaCl and water intakes in NS+mus group

were significantly smaller than those of the NS + NS

group at all time points (P< 0.05), but the bic + mus

group was significantly larger than NS+mus group

(P< 0.05) and was not significantly different from the

NS + NS group (P> 0.05), as shown Fig. 3A, B. From

the two figures, it could also be seen that there was no

significant difference between bic + NS group and

NS+ NS at every time point (P> 0.05).

FLI expression in NTS and PBN induced by CeAmicroinjection of muscimol in sodium-depleted rats

As shown in Fig. 4, few FLI-positive neurons were

observed in caudal NTS (cNTS), intermediate NTS

(iNTS), rostral NTS (rNTS) or PBN in rats of the

control group (Fig. 4A, D, G, J), while the number of

Fig. 1. Photomicrograph showing an example of locations of bilateral injection sites in the CeA. Arrows point to the injection sites within the CeA.

opt, optic tract; BLA, basolateral amygdaloid nucleus; CeA, central nucleus amygdale. Scale bar = 1000 lm.

Fig. 2. Microinjection of muscimol (0.02, 0.2, 0.35 and 0.5 nmol) bilaterally into the CeA dose-dependently decreased the 0.3 M NaCl (A) and water

(B) intake in sodium-depleted rats. ⁄P< 0.05, compared with the saline control group.

280 Q. Wang et al. / Neuroscience 223 (2012) 277–284

FLI-positive cells in the cNTS, iNTS, and LPBN increased

significantly in rats with CeA microinjection of muscimol

(0.2 nmol, in 0.2 ll) (Fig. 4B, E, K). Unpaired t-testsshowed (Fig. 5) that the number of FLI-positive cells in

the cNTS in rats with muscimol microinjection was over

four times more than that of the control group rats

(t= 10.42, P< 0.001). Although statistical analysis

revealed a significant increase in FLI expression in the

iNTS after the muscimol injection (t= 5.99, P< 0.001),

the increase was not as distinct as that in cNTS. The

rats injected with saline and muscimol all had extremely

low FLI expressions in rNTS and their FLI expressions

showed no significant difference (t= 1.06, P> 0.05). In

the PBN, FLI-positive neurons were mostly presented

in the middle part. Distinct FLI expression was induced

in the bilateral external lateral subnucleus (el) and

external medial subnucleus (emx) of the PBN (t= 9.45,

P< 0.001 and t= 6.17, P< 0.001). In the other

subnuclei of the PBN, no significant difference in the FLI

expression was detected between the two groups.

DISCUSSION

The role of GABA receptors in the CeA in mediatingthe sodium intake

Results from the present study have demonstrated that

microinjection of muscimol, a GABAA-receptor agonist,

into the CeA significantly attenuated the 0.3 M NaCl

intake in a dose-dependent manner in the sodium-

depleted rat, and this inhibitory effect was blocked by

bicuculline, a selective GABAA-receptor antagonist,

application to the CeA. This result is not only consistent

with the previous report that the salt intake was inhibited

by CeA lesions (Galaverna et al., 1993; Seeley et al.,

1993), but also suggests that the GABAA receptor in the

Fig. 3. Microinjection of bicuculline bilaterally into the CeA 15 min prior to muscimol (0.2 nmol) injection blocked the intra-CeA muscimol-induced

inhibition of 0.3 M NaCl (A) and water (B) intake in sodium-depleted rats. ⁄P< 0.05, compared with saline (NS + NS) control groups; #P < 0.05,

compared with bicuculline + muscimol (bic + mus) group. Note: Microinjection of bicuculline (i.e., bic + NS) alone into to the CeA had no effect on

0.3 M NaCl (A) and water (B) intake as compared with NS+ NS group (P> 0.05). NS +mus, saline + muscimol.

Q. Wang et al. / Neuroscience 223 (2012) 277–284 281

CeA is involved in the modulation of the sodium intake. We

also found that the bicuculline injection alone into the CeA

had no effect on the sodium intake, suggesting that the

GABAA receptors in the CeA lack a tonic activity on the

sodium intake, and the blocking effect of bicuculline on

the muscimol-induced inhibition of the sodium intake is

not a result of facilitation of the sodium intake. Although

muscimol injection into the CeA inhibited salt and water

intake during the 180-min observation period, the total

intake of the 0.3 M NaCl solution and water remained

unaffected in 24 h of the test, suggesting that muscimol

has a short-term inhibition on the salt and water intake

and that the sodium–water homoeostasis of the body is

not influenced by the acute perturbation of the GABA

system in the amygdala.

Previous studies have indicated that drinking water

can be inhibited by intracerebroventricular or systemic

muscimol in rats with water deprivation (Jones and

Mogenson, 1982; Houston et al., 2002; Tanaka et al.,

2003), but it is unknown where the active location of the

GABA receptors for inhibition of the water intake in the

central nervous system is. The results of this study

provided evidence for this issue showing that muscimol

application to the CeA for activation of GABAA receptors

dose-dependently decreased the water intake during the

180-min observation period in the sodium-depleted rat,

and this inhibitory effect was blocked by intra-CeA

application of the GABAA receptor antagonist bicuculline,

suggesting that the GABAA receptors in the CeA were

involved in mediating the water intake in the sodium-

depleted rat. In our experiment, injection of 0.02 nmol

muscimol into CeA had no effect on water intake.

Houston’s study (Houston et al., 2002) showed that

subcutaneous injection of low dose muscimol (lower than

0.5 mg/kg) has no effect on thirst in water-deprived rats.

This may explain why injection of 0.02 nmol muscimol

into CeA had no significant effect on water intake in our

experiment.

By comparison of sodium and water intake in the

depleted rats, it is revealed that the 0.3 M NaCl intake is

significantly more than the water intake during the 180-

min observation in the 24-h sodium-depleted rat, and in

intra-CeA administration of the muscimol experiment,

although the inhibitory effects on the 0.3 M NaCl and

water intake both are dose-dependent, the smallest

dose (0.02 nmol) of muscimol significantly inhibits the

sodium intake, but did not influence the water intake.

Moreover, the inhibitory effect of other larger doses

(0.2–0.5 nmol) of muscimol on the water intake did not

occur in the first 15 min (partly in the 30 min, see

Fig. 2B). These facts suggest that the rat with sodium

depletion predominantly selected the sodium intake, and

the decreased sodium intake induced by activation of

the GABAA receptors in the CeA may be a specific

function relative to water intake. However, there is

evidence that CeA lesions or injection of muscimol could

relieve starvation or reduced food intake in food-

deprived rats (Box and Mogenson, 1975; Minano et al.,

1992), suggesting that the GABAA receptor in the CeA

is also involved in mediating the food intake. Therefore,

the function of the CeA in the feeding behavior may be

complicated requiring further investigation.

The possible mechanism of NTS and PBN in CeAGABAA receptor-mediated sodium intake

Substantial neuroanatomical studies have shown that

reciprocal connections exist among the CeA, PBN and

NST, which are parts of the neural system involved in

gustatory and viscerosensory transmissions (Voshart

and van der Kooy, 1981; Halsell, 1998; Whitehead

et al., 2000; Jia et al., 2005). The cNTS neurons receive

the visceral afferent information, via the LPBN, project

to CeA and other forebrain areas, while the rNST

neurons receive the taste information, via MPBN,

project to the CeA (Norgren and Leonard, 1971;

Hamilton and Norgren, 1984; Yamamoto et al., 1997).

The LPBN neurons also receive the visceral post-

ingestion information from area postrema (AP)/medial

NTS (mNTS), and project to the CeA. It has been

known that activation of the AP/mNTS–LPBN pathway

inhibits sodium intake (Ohman and Johnson, 1986;

Fig. 4. FLI expressions in cNST (A, B), iNTS (D, E), rNTS (G, H) and PBN (J, K) following bilateral CeA microinjection of muscimol in sodium-

depleted rats (B, E, H, K) and control rats (A, D, G, J). C, F, I and L are schematics of cNTS, iNTS, rNTS and PBN, respectively. cc, central canal;

NTS, nucleus tractus solitaril; 4V, fourth ventricle. The parabrachial nucleus includes the following six regions: vl, ventral lateral subnucleus; dl,

dorsal lateral subnucleus; el, external lateral subnucleus; cl, central lateral subnucleus; m, medial subnucleus; and exm, extreme medial

subnucleus. SCP, superior cerebellar peduncle. Scale bar = 100 lm.

282 Q. Wang et al. / Neuroscience 223 (2012) 277–284

Menani et al., 1998; de Oliveira et al., 2007; David et al.,

2008), while lesion of this pathway enhanced sodium and

water intake in the sodium-depleted rats induced by acute

and chronic sodium depletion (Contreras and Stetson,

1981; Ogihara et al., 2009). Importantly, The CeA not

only receives the ascending projection from the

brainstem of NTS and PBN, but also contains neurons

that descendingly project to the NTS and PBN (Voshart

and van der Kooy, 1981; Halsell, 1998; Whitehead

et al., 2000; Jia et al., 2005) and modulates the

brainstem taste neuronal activity in the ingestion

behavior (Li et al., 2002, 2005; Kang et al., 2004). As

reported by Andrade-Franze et al. (2010), activation of

the CeA has a facilitating effect on sodium appetite. The

results of this study indicated that microinjection of

muscimol into the CeA for inhibition of the neuronal

activity in this region increased FLI expression in the

cNTS, iNTS and LPBN in the sodium-depleted rats,

suggesting that activation of the GABAA receptors in the

CeA may depress the inhibitory action (disinhibition) of

CeA on the LPBN neurons leading to activation of the

AP/mNTS–LPBN descending inhibitory pathway.

In addition, the external lateral subnucleus of PBN (el-

PBN) receives the nociceptive afferent information from

the viscera (Yamamoto and Sawa, 2000) and is related

to negative hedonics or ingestive behavior, while the

dorsal lateral subnucleus (dl-PBN) is related to positive

hedonics or ingestive behavior (Yamamoto et al., 1994).

For example, intraperitoneal injection of LiCl results in

an increased number of FLI-positive neurons in the el-

PBN associated with a nociceptive aversive response,

but no observable FLI-positive neurons in the dl-PBN

(Yamamoto et al., 1992; Sakai and Yamamoto, 1997).

In the present study, microinjection of muscimol into the

CeA in the sodium-depleted rats markedly increased the

number of FLI-positive neurons in the el-PBN, but not in

Fig. 5. The numbers of FLI neurons in the NTS and the PBN

subnucleus after bilateral CeA microinjection of muscimol in sodium-

depleted rats. ⁄⁄⁄P< 0.001, compared with saline control group.

cNTS, the caudal NTS; iNTS, the intermediate NTS; rNTS, the rostral

NTS. Five regions of the parabrachial nucleus are as follows: vl,

ventral lateral subnucleus; dl, dorsal lateral subnucleus; el, external

lateral subnucleus; exm, extreme medial subnucleus; m, medial

subnucleus.

Q. Wang et al. / Neuroscience 223 (2012) 277–284 283

the dl-PBN, implying that following activation of the

GABAA receptors in the CeA, the neurons related to

negative hedonics or aversive behavior in the el-PBN

decrease sodium intake in sodium-depleted rats.

CONCLUSION

In conclusion, the results of the present study suggest

that the GABAA receptor in the CeA is involved in

inhibiting the sodium depletion-induced sodium intake.

This inhibitory effect may be produced by the activation

of GABAA receptor in the CeA, in which it depresses the

inhibitory action (disinhibition) of the CeA on the NTS

and PBN neurons leading to activation of the NTS and

PBN-descending inhibitory pathways which depress the

sodium intake behavior induced by sodium depletion.

Acknowledgments—The authors wish to thank Dr. F.Q. Huo and

Y.X. Zhu for valuable technical advice, and thank Dr. J.S. Tang

for valuable comments on this manuscript. The present work

was supported by the National Natural Science Foundation of

China (Nos. 31171052 and 30970973).

REFERENCES

Abe M, Tokunaga T, Yamada K, Furukawa T (1988) Gamma-

aminobutyric acid and taurine antagonize the central effects of

angiotensin-II and renin on the intake of water and salt, and on

blood-pressure in rats. Neuropharmacology 27:309–318.

Andrade-Franze GM, Andrade CA, De Luca Jr LA, De Paula PM,

Menani JV (2010) Lateral parabrachial nucleus and central

amygdala in the control of sodium intake. Neuroscience

165:633–641.

Andrade CA, Barbosa SP, De Luca Jr LA, Menani JV (2004)

Activation of alpha2-adrenergic receptors into the lateral

parabrachial nucleus enhances NaCl intake in rats.

Neuroscience 129:25–34.

Araki T, Kiyama H, Tohyama M (1992) The Gaba(a) receptor-

gamma-1 subunit is expressed by distinct neuronal populations.

Mol Brain Res 15:121–132.

Berridge KC, Flynn FW, Schulkin J, Grill HJ (1984) Sodium depletion

enhances salt palatability in rats. Behav Neurosci 98:652–660.

Box BM, Mogenson GJ (1975) Alterations in ingestive behaviors after

bilateral lesions of the amygdala in the rat. Physiol Behav

15:679–688.

Callera JC, Oliveira LB, Barbosa SP, Colombari DS, De Luca Jr LA,

Menani JV (2005) GABA(A) receptor activation in the lateral

parabrachial nucleus induces water and hypertonic NaCl intake.

Neuroscience 134:725–735.

Cho YK, Li CS, Smith DV (2003) Descending influences from the

lateral hypothalamus and amygdala converge onto medullary

taste neurons. Chem Senses 28:155–171.

Contreras RJ, Stetson PW (1981) Changes in salt intake lesions of

the area postrema and the nucleus of the solitary tract in rats.

Brain Res 211:355–366.

David RB, Menani JV, De Luca LA (2008) Serotonergic receptor

blockade in the lateral parabrachial nucleus: different effects on

hypertonic and isotonic NaCl intake. Brain Res 1187:137–145.

de Oliveira LB, Callera JC, De Luca Jr LA, Colombari DS, Menani JV

(2007) GABAergic mechanisms of the lateral parabrachial

nucleus on sodium appetite. Brain Res Bull 73:238–247.

Galaverna O, De Luca Jr LA, Schulkin J, Yao SZ, Epstein AN (1992)

Deficits in NaCl ingestion after damage to the central nucleus of

the amygdala in the rat. Brain Res Bull 28:89–98.

Galaverna OG, Seeley RJ, Berridge KC, Grill HJ, Epstein AN,

Schulkin J (1993) Lesions of the central nucleus of the amygdala:

1. Effects on taste reactivity, taste-aversion learning and sodium

appetite. Behav Brain Res 59:11–17.

Geerling JC, Engeland WC, Kawata M, Loewy AD (2006)

Aldosterone target neurons in the nucleus tractus solitarius drive

sodium appetite. J Neurosci 26:411–417.

Geerling JC, Loewy AD (2006) Aldosterone-sensitive neurons in the

nucleus of the solitary tract: bidirectional connections with the

central nucleus of the amygdala. J Comp Neurol 497:646–657.

Halsell CB (1998) Differential distribution of amygdaloid input across

rostral solitary nucleus subdivisions in rat. Ann N Y Acad Sci

855:482–485.

Hamilton RB, Norgren R (1984) Central projections of gustatory

nerves in the rat. J Comp Neurol 222:560–577.

Houston AJ, Wong JCL, Ebenezer IS (2002) Effects of subcutaneous

administration of the gamma-aminobutyric acid(A) receptor

agonist muscimol on water intake in water-deprived rats. Physiol

Behav 77:445–450.

Huang T, Yan J, Kang Y (2003) Role of the central amygdaloid

nucleus in shaping the discharge of gustatory neurons in the rat

parabrachial nucleus. Brain Res Bull 61:443–452.

Jia HG, Zhang GY, Wan Q (2005) A GABAergic projection from the

central nucleus of the amygdala to the parabrachial nucleus: an

ultrastructural study of anterograde tracing in combination with

post-embedding immunocytochemistry in the rat. Neurosci Lett

382:153–157.

Jones DL, Mogenson GJ (1982) Central injections of spiperone and

GABA: attenuation of angiotensin II stimulated thirst. Can J

Physiol Pharmacol 60:720–726.

Kang Y, Yan J, Huang T (2004) Microinjection of bicuculline into the

central nucleus of the amygdala alters gustatory responses of the

rat parabrachial nucleus. Brain Res 1028:39–47.

Kimura EH, Oliveira LB, Colombari DSA, De Luca LA, Menani JV,

Callera JC (2008) Sodium intake by hyperosmotic rats treated

with a GABA(A) receptor agonist into the lateral parabrachial

nucleus. Brain Res 1190:86–93.

Li CS, Cho YK, Smith DV (2002) Taste responses of neurons in the

hamster solitary nucleus are modulated by the central nucleus of

the amygdala. J Neurophysiol 88:2979–2992.

Li CS, Cho YK, Smith DV (2005) Modulation of parabrachial taste

neurons by electrical and chemical stimulation of the lateral

hypothalamus and amygdala. J Neurophysiol 93:1183–1196.

284 Q. Wang et al. / Neuroscience 223 (2012) 277–284

McDonald AJ (1998) Cortical pathways to the mammalian amygdala.

Prog Neurobiol 55:257–332.

McDonald AJ, Augustine JR (1993) Localization of GABA-like

immunoreactivity in the monkey amygdala. Neuroscience

52:281–294.

Menani JV, Colombari DS, Beltz TG, Thunhorst RL, Johnson AK

(1998) Salt appetite: interaction of forebrain angiotensinergic and

hindbrain serotonergic mechanisms. Brain Res 801:29–35.

Minano FJ, Sancho MSM, Sancibrian M, Salinas P, Myers RD (1992)

Gaba(a) receptors in the amygdala – role in feeding in fasted and

satiated rats. Brain Res 586:104–110.

Nitecka L, Ben-Ari Y (1987) Distribution of GABA-like immunore-

activity in the rat amygdaloid complex. J Comp Neurol 266:45–55.

Norgren R, Leonard CM (1971) Taste pathways in rat brainstem.

Science 173:1136–1139.

Ogihara CA, Schoorlemmer GHM, Colombari E, Sato MA (2009)

Changes in sodium appetite evoked by lesions of the commissural

nucleus of the tractus solitarius. Braz J Med Biol Res 42:561–566.

Ohman LE, Johnson AK (1986) Lesions in lateral parabrachial

nucleus enhance drinking to angiotensin II and isoproterenol. Am

J Physiol 251:R504–R509.

Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates.

5th ed. San Diego, CA: Academic Press.

Prakash MR, Norgren R (1991) Comparing salt appetites: induction

with intracranial hormones or dietary sodium restriction. Brain Res

Bull 27:397–401.

Saha S, Batten TF, Henderson Z (2000) A GABAergic projection from

the central nucleus of the amygdala to the nucleus of the solitary

tract: a combined anterograde tracing and electron microscopic

immunohistochemical study. Neuroscience 99:613–626.

Sakai N, Yamamoto T (1997) Conditioned taste aversion and c-fos

expression in the rat brainstem after administration of various

USs. Neuroreport 8:2215–2220.

Seeley RJ, Galaverna O, Schulkin J, Epstein AN, Grill HJ (1993)

Lesions of the central nucleus of the amygdala: II. Effects on

intraoral NaCl intake. Behav Brain Res 59:19–25.

Sun N, Cassell MD (1993) Intrinsic Gabaergic neurons in the rat

central extended amygdala. J Comp Neurol 330:381–404.

Tanaka J, Fujisawa S, Nomura M (2003) GABAergic modulation of

the ANG II-induced drinking response in the rat medial preoptic

nucleus. Pharmacol Biochem Behav 76:43–51.

Unger T, Bles F, Ganten D, Lang RE, Rettig R, Schwab NA (1983)

Gabaergic stimulation inhibits central actions of angiotensin-II –

pressor-responses, drinking and release of vasopressin. Eur J

Pharmacol 90:1–9.

Vendramini RC, Pereira DTB, Borella TL, Menani JV, De Luca LA

(2009) Damage to the central amygdala produces differential

encephalic c-fos expression in the water deprivation-partial

rehydration protocol. Brain Res 1304:80–89.

Voshart K, van der Kooy D (1981) The organization of the efferent

projections of the parabrachial nucleus of the forebrain in the rat: a

retrograde fluorescent double-labeling study. Brain Res

212:271–286.

Weisinger RS, Considine P, Denton DA, Leksell L, McKinley MJ,

Mouw DR, Muller AF, Tarjan E (1982) Role of sodium

concentration of the cerebrospinal fluid in the salt appetite of

sheep. Am J Physiol 242:R51–R63.

Whitehead MC, Bergula A, Holliday K (2000) Forebrain projections to

the rostral nucleus of the solitary tract in the hamster. J Comp

Neurol 422:429–447.

Yamamoto T, Sawa K (2000) C-Fos-like immunoreactivity in the

brainstem following gastric loads of various chemical solutions in

rats. Brain Res 866:135–143.

Yamamoto T, Sako N, Sakai N, Iwafune A (1997) Gustatory and

visceral inputs to the amygdala of the rat: conditioned taste

aversion and induction of c-fos-like immunoreactivity. Neurosci

Lett 226:127–130.

Yamamoto T, Shimura T, Sakai N, Ozaki N (1994) Representation of

hedonics and quality of taste stimuli in the parabrachial nucleus of

the rat. Physiol Behav 56:1197–1202.

Yamamoto T, Shimura T, Sako N, Azuma S, Bai WZ, Wakisaka S

(1992) C-fos expression in the rat brain after intraperitoneal

injection of lithium chloride. Neuroreport 3:1049–1052.

Zardetto-Smith AM, Beltz TG, Johnson AK (1994) Role of the central

nucleus of the amygdala and bed nucleus of the stria terminalis in

experimentally-induced salt appetite. Brain Res 645:123–134.

(Accepted 31 July 2012)(Available online 9 August 2012)