Activation of μ-opioid receptors in the central nucleus of the amygdala induces hypertonic sodium intake

Neuroscience 233 (2013) 28–43

ACTIVATION OF l-OPIOID RECEPTORS IN THE CENTRAL NUCLEUS OF THE AMYGDALA INDUCES HYPERTONIC SODIUM INTAKE JUNBAO YAN,   JINRONG LI,   JIANQUN YAN, * HUILING SUN, QIAN WANG, KE CHEN, BO SUN, XIAOJING WEI, LIN SONG, XIAOLIN ZHAO, SHUANGYU WEI AND LING HAN

Key words: sodium appetite, water intake, DAMGO, CTAP.

Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an Jiaotong University College of Medicine, 76# W. Yanta Road, Xi’an, Shaanxi 710061, PR China

INTRODUCTION +

Sodium (Na ), as the most abundant electrolyte in the extracellular fluid (ECF) compartment, plays a critical role in maintaining hydromineral and cardiovascular homeostasis. Na+ hunger, which is also known as salt appetite, is a goal-directed behavior that arises in rodents and other species in specific response to volume and/or Na+ depletion in the ECF compartment (Weisinger et al., 1979; Johnson and Thunhorst, 1997). The amygdala, a brain structure containing distinct nuclei, influences a great number of behaviors tailored to promote the animal’s adaptation to external and internal stimuli (Price et al., 1987), and has been believed to participate in the regulation of sodium intake (Covian et al., 1975; Galaverna et al., 1992; Zhang et al., 1993; Zardetto-Smith et al., 1994; Swanson and Petrovich, 1998; Johnson et al., 1999). The distinct nuclei within the amygdala may play specific functional roles in the control of salt intake. Surgical lesions of the basolateral amygdala (BLA) inhibit salt intake induced by mineralocorticoid treatment (Nachman and Ashe, 1974). Lesions of the medial amygdala (MeA) impair mineralocorticoid-induced salt intake but do not affect salt intake promoted by sodium depletion (Nitabach et al., 1989; Zhang et al., 1993). Different studies have also shown the importance of the central nucleus of the amygdala (CeA) in the control of the ingestion of sodium intake. Bilateral electrolytic lesions of the CeA reduce spontaneous sodium intake, as well as sodium appetite induced by subcutaneous injections of the mineralocorticoid deoxycorticosterone, the a2-adrenoceptor antagonist yohimbine, or angiotensin II (ANG II), intracerebral ventricle injections of renin or by 24 h of sodium depletion in rats treated with furosemide (Galaverna et al., 1992; Zardetto-Smith et al., 1994). However, water intake induced by subcutaneous injections of ANG II or by cellular dehydration is not affected by lesions in the CeA, reinforcing the concept that lesions of the CeA reduce specifically sodium appetite (Zardetto-Smith et al., 1994). Pharmacological activation of 5-HT3 receptors located within the CeA inhibits salt intake in sodium-depleted rats (Luz et al., 2007). Lately, our laboratory has also demonstrated that the activation of GABAA receptors in the CeA inhibits sodium intake in sodium-depleted rats, suggesting an

Abstract—Opioid mechanisms are involved in the control of water and NaCl intake and opioid receptors (ORs) are present in the central nucleus of the amygdala (CeA), a site of important facilitatory mechanisms related to the control of sodium appetite. Therefore, in the present study we investigated the effects of the activation of l-ORs in the CeA on 0.3 M NaCl and water intake in rats. Male Sprague–Dawley rats with stainless steel cannulas implanted bilaterally in the CeA were used. In rats submitted to water deprivation– partial rehydration, bilateral injections of the selective l-OR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) in the doses of 1, 2, and 4 nmol into the CeA induced a dose-related increase of 0.3 M NaCl intake and water intake, and bilateral injections of the selective l-OR antagonist D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) in the doses of 0.5, 1, and 2 nmol into the CeA produced a dose-related decrease of 0.3 M NaCl and water intake induced by DAMGO 2 nmol into the same site. In rats treated with the diuretic furosemide (10 mg/kg b.w.) combined with the angiotensin-converting enzyme inhibitor captopril (5 mg/kg b.w.) injected subcutaneously, bilateral injections of DAMGO 2 nmol into the CeA increased 0.3 M NaCl intake and water intake and the blockade of l-ORs with CTAP 1 nmol injected into the CeA reduced the increase in 0.3 M NaCl intake and water intake induced by DAMGO 2 nmol into the same site. Bilateral injections of DAMGO into the CeA did not change urinary volume, sodium urinary excretion and mean arterial pressure, but increased activity. Thus stimulating l-ORs in the CeA increases hypertonic sodium intake, whereas antagonizing these sites inhibits hypertonic sodium intake. Together, our results implicate l-ORs in the CeA in a positive regulation of sodium intake. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

*Corresponding author. Tel/fax: +86-2982655199. E-mail address: [email protected] (Jianqun Yan).   These authors contributed equally to this work. Abbreviations: ANG II, angiotensin II; ANOVA, analysis of variance; BLA, basolateral amygdala; CAP, captopril; CeA, central nucleus of the amygdala; CTAP, D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2; DAMGO, [DAla2, N-Me-Phe4, Gly5-ol]-enkephalin; ECF, extracellular fluid; FURO, furosemide; HSD2, hydroxysteroid dehydrogenase type 2; LPBN, lateral parabrachial nucleus; MAP, mean arterial pressure; NTS, nucleus of the solitary tract; ORs, opioid receptors; Na+, sodium; WD, water-deprived rat.

0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.12.026 28

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important facilitatory mechanism related to the control of sodium appetite in the CeA (Wang et al., 2012). It is well accepted that opioid mechanisms play an important role in the control of ingestive behavior and specifically on the ingestion of sodium and water (Cooper and Gilbert, 1984; Hubbell and McCutcheon, 1993; Lucas et al., 2007; De Oliveira et al., 2008). Opioid receptors (ORs) are present in the CeA (Mansour et al., 1995; Poulin et al., 2006; Glass et al., 2009) and the CeA contains intrinsic neurons and axon terminals that contain opioid peptides (Fallon and Leslie, 1986; Cassell and Gray, 1989; Poulin et al., 2006). Most of neurons in the rat CeA are inhibited by the activation of l-ORs located within the CeA (Zhu and Pan, 2004; Chieng et al., 2006). Recently, Grondin et al. (2011) found that combining in situ hybridization (against enkephalin and l-OR mRNA) and immunohistochemistry (against Fos) revealed a hypovolemia-induced Fos expression in the enkephalinergic subpopulations of the CeA, and hypovolemia also induced transient Fos expression in l-OR-expressing neurons in the CeA, suggesting the enkephalin/l-OR system is a putative facilitator of Na+ intake in the CeA. Although previous studies had reported the involvement of different neurotransmitters and receptors in the CeA in the control of sodium intake, no study investigated the participation of opioid mechanisms in the CeA in the control of sodium and water intake. It has been demonstrated that water deprivation– partial rehydration (WD–PR protocol) induces sodium appetite (Sato et al., 1996; De Luca et al., 2002). Pereira et al. (2010b) also found that the WD–PR induced c-Fos immunoreactivity in the rat CeA. In the WD–PR protocol, the water-deprived rat (WD) is first allowed to drink only water until it satiates its thirst (PR). Then, the PR is immediately followed by access to another bottle containing NaCl solution (sodium appetite test). The ingestion of hypertonic NaCl solution in this context can be considered as an expression of sodium appetite, because it results from negative sodium balance and persistent hypovolemia (Weisinger et al., 1985; Sato et al., 1996; Johnson, 2007; Pereira et al., 2010b). In addition, different studies have also demonstrated that subcutaneous injections of the diuretic furosemide (FURO, 10 mg/kg b.w.) combined with the angiotensinconverting enzyme inhibitor captopril (CAP, 5 mg/ kg b.w.) (FURO/CAP protocol) induces sodium appetite (Thunhorst and Johnson, 1994; Menani et al., 1996; Pereira et al., 2010a; Da Silva et al., 2011). Therefore, considering the presence of ORs and neurotransmitters in the CeA and the already reported role of the CeA and central opioid mechanisms in the control of water and sodium intake, in the present study we investigated the effects of bilateral injections of [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) (the selective l-OR agonist) and D-Phe-Cys-Trp-Arg-ThrPen-Thr-NH2 (CTAP) (the selective l-OR antagonist) alone or combined into the CeA in the control of 0.3 M NaCl and water intake in rats submitted to the WD–PR or in FURO + CAP-treated rats and the effects of

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DAMGO injected into the CeA on water deprivationinduced water intake.

EXPERIMENTAL PROCEDURES Animals A total of 109 adult male Sprague–Dawley rats weighing 290 ± 20 g were used in the present study. The rats were housed in individual stainless steel cages (before cerebral cannulas) and metabolism cages (after cerebral cannulas) with free access to pelleted laboratory rodent chow, distilled water and 0.3 M NaCl solution. Rats were maintained at a colony room temperature of 23 ± 2°C, humidity of 55 ± 10% and on a 12-h light/dark cycle with light onset at 7:00 AM. The experimental protocols followed the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, 1996). All efforts were made to reduce animal discomfort and the number of animals used.

Cerebral cannulas Following anesthesia with an intraperitoneal dose of chloral hydrate (300 mg/kg b.w.), the rats were secured in a stereotaxic apparatus (SN-2N, Narishige Group, Tokyo, Japan) for bilateral implantation of stainless steel cannulas (23 gauge) into the CeA. The stereotaxic coordinates of the CeA were determined according to the brain atlas of the rat (Paxinos and Watson, 1997) and were: 2.3 mm posterior to bregma, 4.0 mm lateral to the midline suture, and 7.0 mm below the skull surface. The tips of the cannulas were placed 1 mm above the CeA. The cannulas were cemented to the skull bone with dental acrylic resin and jeweler screws and filled with obstructors (30 gauge). After the cerebral surgery, the rats were allowed to recover for 7 days in individual metabolism/ feeding–drinking cages with free access to pelleted laboratory rodent chow, distilled water and 0.3 M NaCl solution before starting ingestion tests.

Injections into the CeA Bilateral injections into the CeA were administered using 1-lL Hamilton syringes (Hamilton, Reno, NV, USA) connected by PE-10 polyethylene tubing to 30-gauge injection cannulas. At the time of testing, obturators were removed and the injection cannula (1 mm longer than the guide cannula) was carefully inserted into the guide cannula, and manual injection was initiated 15 s later. The injection volume into the CeA was 0.5 lL in each site and the injection was delivered at a flow rate of 0.5 lL/min. The injection cannulas were maintained in place for 30 s after delivery of the drug or vehicle to minimize the backflow. The obturators were replaced after the injections, and the rats were placed back into their metabolism cages.

Drugs The drugs including the selective l-OR agonist DAMGO, the highly selective l-OR antagonist CTAP, the diuretic FURO, and the angiotensin-converting enzyme inhibitor CAP, were purchased from Sigma–Aldrich (Sigma–Aldrich, Saint Louis, MO, USA). All the drugs were dissolved in sterile 0.9% (w/v) saline solution. Accordingly, the 0.9% saline solution was used as vehicle. The drugs and vehicle solutions were made just before the infusion. The drugs injected into the CeA were DAMGO at the doses of 1 nmol, 2 nmol, 4 nmol/0.5 lL, and CTAP at the doses of 0.5 nmol, 1 nmol, 2 nmol/0.5 lL. FURO and CAP were administered subcutaneously at 10 and 5 mg/

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kg b.w., respectively as described previously (Thunhorst and Johnson, 1994; Menani et al., 1996; Pereira et al., 2010a; Da Silva et al., 2011).

0.3 M NaCl and water intake by rats submitted to the WD–PR The WD–PR protocol is a sequence of water deprivation and partial volume repletion ensured by water intake that precedes the sodium appetite test. In the present study, rats were deprived of water and 0.3 M NaCl solution, with free access to food for 24 h (WD) (from 08:00 to 08:00). Then, food was removed and water was offered for 2 h (PR). The rats were tested in their individual metabolism/feeding–drinking cages, a part of feeding– drinking–activity analyser (Cat. No. 41800111213) (UGO Basline Biological Research Apparatus, COMERIO-Varese, ITALY). Food was not available for the rats during tests. Experiment 1, at first, bilateral-cannulated rats (n = 16) not submitted to the WD–PR receiving CeA injections of isotonic saline solution were performed, just to show the difference in 0.3 M NaCl and water intake presented by drug-free WD–PR  no WD–PR rats. Following the CeA injections, cumulative 0.3 M NaCl and water intakes were automatically recorded by the feeding–drinking–activity analyser at 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min (two-bottle test). Three days after the test, the rats, following the WD–PR, received bilateral injections of DAMGO in several doses (1, 2 and 4 nmol) or isotonic saline solution into the CeA. The rats were randomly assigned to treatment groups to receive either (a) saline, (b) DAMGO 1 nmol, (c) DAMGO 2 nmol, or (d) DAMGO 4 nmol. Each rat received all four treatments in a counter-balanced design. Following the CeA injections, 0.3 M NaCl was offered, and cumulative 0.3 M NaCl and water intakes were automatically recorded by the feeding–drinking–activity analyser at 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min (two-bottle test). A recovery period of at least 3 days was allowed between tests. Experiment 2, water intake after bilateral injections of DAMGO (2 nmol) or saline into the CeA was tested in rats that had access only to water during tests. Following the WD–PR, bilateralcannulated rats (n = 12) received CeA injections of DAMGO (2 nmol) or saline. The rats were randomly assigned to treatment groups to receive either (a) saline, or (b) DAMGO 2 nmol. Each rat received both treatments in a counter-balanced design. Following the CeA injections, 0.3 M NaCl was not offered and only cumulative water intake was automatically recorded by the feeding–drinking–activity analyser at 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min (one-bottle test). A recovery period of at least 3 days was allowed between tests. Experiment 3, following the WD–PR, bilateral-cannulated rats (n = 15) received CeA injections of combinations of saline, DAMGO (2 nmol), and CTAP (0.5, 1 and 2 nmol). The rats were randomly assigned to treatment groups to receive either (a) saline + saline, (b) saline + DAMGO 2 nmol, (c) CTAP 0.5 nmol + DAMGO 2 nmol, (d) CTAP 1 nmol + DAMGO 2 nmol, or (e) CTAP 2 nmol + DAMGO 2 nmol. Each rat received all five treatments in a counter-balanced design. Bilateral injections of CTAP or saline into the CeA were performed 10 min before the injections of DAMGO or saline into the CeA. Following the CeA injections, 0.3 M NaCl was offered, and cumulative 0.3 M NaCl and water intakes were automatically recorded by the feeding–drinking–activity analyser at 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min (two-bottle test). A recovery period of at least 3 days was allowed between tests.

0.3 M NaCl and water intake by FURO + CAP-treated rats The FURO/CAP protocol is that a subcutaneous injection of the diuretic FURO (10 mg/kg b.w.) is combined with an angiotensinconverting enzyme inhibitor CAP (5 mg/kg b.w.). In the present

study, rats received subcutaneous injections of the diuretic FURO (10 mg/kg b.w.) plus CAP (5 mg/kg b.w.) at 08: 00 and then were returned to their individual metabolism/feeding– drinking cages in the absence of food, water and 0.3 M NaCl solution. The rats were tested in their individual metabolism/ feeding–drinking cages. Food was not available for the rats during tests. Experiment 1, at first, bilateral-cannulated rats (n = 12) treated with subcutaneous injections of isotonic saline solution (1 ml/100 g b.w.) instead of the FURO plus CAP receiving CeA injections of isotonic saline solution were performed, just to show the difference in 0.3 M NaCl and water intake presented by drugfree FURO/CAP  no FURO/CAP rats. One hour after subcutaneous injections of isotonic saline solution, the rats received bilateral injections of isotonic saline solution into the CeA. Following the CeA injections, water and 0.3 M NaCl, but no food, were available for rats and cumulative intakes of 0.3 M NaCl and water were automatically measured by the feeding– drinking–activity analyser at 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min (two-bottle test). Three days after the test, the rats received subcutaneous injections of the FURO (10 mg/ kg b.w.) plus CAP (5 mg/kg b.w.). One hour after the FURO + CAP treatment, the rats received bilateral injections of combinations of saline, DAMGO (2 nmol) and CTAP (1 nmol) into the CeA. The rats were randomly assigned to treatment groups to receive either (a) saline + saline, (b) saline + DAMGO 2 nmol, (c) CTAP 1 nmol + saline, or (d) CTAP 1 nmol + DAMGO 2 nmol. Each rat received all four treatments in a counter-balanced design. Bilateral injections of CTAP or saline into the CeA were performed 10 min before the injections of DAMGO or saline into the same site. The order of treatments was randomized also because repeated FURO + CAP injections enhances stimulated and spontaneous NaCl intake (Pereira et al., 2010a). Following the CeA injections, water and 0.3 M NaCl, but no food, were available for rats and cumulative intakes of 0.3 M NaCl and water were automatically measured by the feeding–drinking–activity analyser at 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min (two-bottle test). A recovery period of at least 3 days was allowed between tests. Experiment 2, water intake after bilateral injections of DAMGO (2 nmol) or saline into the CeA was tested in rats that had access only to water during tests. Bilateral-cannulated rats (n = 12) received subcutaneous injections of the diuretic FURO (10 mg/kg b.w.) plus CAP (5 mg/kg b.w.). One hour after the FURO + CAP treatment, the rats received bilateral injections of DAMGO (2 nmol) or saline into the CeA. The rats were randomly assigned to treatment groups to receive either (a) saline, or (b) DAMGO. Each rat received both treatments in a counter-balanced design. Following the CeA injections, only water, but no 0.3 M NaCl and food, was available for rats and cumulative water intake was automatically measured by the feeding–drinking–activity analyser at 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min (one-bottle test). A recovery period of at least 3 days was allowed between tests.

Water intake by water-deprived rats Dehydration that derives from water deprivation induces one of the most powerful behavioral drives known as thirst. Thirst leads to the selective ingestion of water, a behavior vital for most terrestrial vertebrates to replace their water loss and correct dehydration (Epstein, 1990). To investigate whether l-ORs located within the CeA participated in the regulation of water intake in rats, we examined the effects of bilateral injections of DAMGO into the CeA on water intake in waterdeprived rats. The rats were tested in their individual metabolism/feeding–drinking cages. Food and 0.3 M NaCl solution were not available for the rats during tests. At first, bilateral-cannulated rats (n = 12) not submitted to water deprivation receiving CeA injections of isotonic saline solution were performed, just to show the difference in water

J. Yan et al. / Neuroscience 233 (2013) 28–43 intake presented by drug-free dehydrated  normohydrated rats. Following the CeA injections, only water, but no 0.3 M NaCl and food, was available for rats and cumulative water intake was automatically recorded by the feeding–drinking–activity analyser at every 15 min during 240 min. Three days after the test, the rats, following 14 h of water deprivation (both distilled water and 0.3 M NaCl solution were removed from 18:00 to 08:00 the night before the experiment), were randomly assigned to treatment groups to receive CeA injections of DAMGO in

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several doses (1, 2 and 4 nmol) or isotonic saline solution (control) in a counter-balanced design. Following the CeA injections, only water, but no 0.3 M NaCl and food, was available for rats and cumulative water intake was automatically recorded by the feeding–drinking–activity analyser at every 15 min during 240 min. A recovery period of at least 3 days was allowed between tests.

Renal excretion Bilateral-cannulated rats (n = 10) were randomly assigned to treatment groups to received CeA injections of DAMGO (2 nmol) or isotonic saline solution (control) in a counterbalanced design. Following the CeA injections, urine samples were collected by their individual metabolism/feeding–drinking cages at every 30 min during 240 min. Urinary sodium concentration was measured using a digital flame photometer (Model 410, SHERWOOD, England). During the tests of renal excretion, water, 0.3 M NaCl solution and food were not available for the rats.

Arterial pressure recording Mean arterial pressure (MAP) was measured using a noninvasive sphygmomanometer (BP-98A, Softron, Japan). To minimize the stress during the measurements, bilateralcannulated rats (n = 10) were accustomed to the process of the noninvasive measurement of arterial pressure once a day for 3 days. After the adaptive phase, the rats were randomly assigned to treatment groups to receive CeA injections of DAMGO (2 nmol) or isotonic saline solution (control) in a counter-balanced design. Following the CeA injections, MAP was recorded using the non-invasive sphygmomanometer at every 30 min during 240 min. During arterial pressure recording, water, 0.3 M NaCl solution and food were not available for the rats.

Activity recording The rats were tested in their individual metabolism/feeding– drinking cages. The coordinate ambulatory activity and rearing of the rodent on test can be measured via the optional infrared motion detector mounted on the feeding–drinking–activity analyser. Bilateral-cannulated rats (n = 10) were randomly assigned to treatment groups to received CeA injections of

Fig. 1. Diagram based on the Paxinos and Watson Atlas showing sequential coronal sections of areas reached by the injections within (hachured area) and outside (dotted area) the central nucleus of the amygdala. OT, optic tract.

Fig. 2. Photomicrograph showing the sites of injections into the CeA (arrows). OT, optic tract; CT, cannula tract; CeA, the central nucleus of the amygdala. Magnification: 1.25.

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Fig. 3. (A) Cumulative 0.3 M NaCl intake; (B) Cumulative water intake by rats submitted to the WD–PR that received bilateral injections of several doses of DAMGO in sites outside the CeA (misplaced injections).

DAMGO (2 nmol) or isotonic saline solution (control) in a counterbalanced design. Following the CeA injections, horizontal activity and vertical activity counts were automatically recorded by the feeding–drinking–activity analyser at every 15 min during 240 min. During activity recording, water, 0.3 M NaCl solution and food were not available for the rats.

a freezing microtome, and analyzed under a light microscope to confirm the injection sites in the CeA with reference to the atlas of Paxinos and Watson.

Statistical analysis Histology After the completion of each experiment, the rats received bilateral injections of 0.5 lL of 2% Chicago sky blue solution into the CeA. The rats were then deeply anesthetized with a high dose of chloral hydrate and perfused transcardially with saline followed by 10% formalin. The brains were removed, fixed in 10% formalin, cut into 40-lm serial coronal sections on

Statistical analysis was performed using SPSS for Windows (version 13.0). Data are presented as means ± standard error of the mean (SEM) and were analyzed for the main effects of treatment and time by appropriate (i.e., 1-way, 2-way) repeated-measures analysis of variance (ANOVA). Post hoc comparisons were by Student–Newman–Keuls multiple comparison test. The statistical significance was set at P less than 0.05.

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Fig. 4. (A) Cumulative 0.3 M NaCl intake; (B) Cumulative water intake by FURO + CAP-treated rats that received bilateral injections of combinations of saline, DAMGO 2 nmol and CTAP 1 nmol in sites outside the CeA (misplaced injections).

RESULTS Histological analysis Fig. 1 corresponds to a diagram based on the Paxinos and Watson Atlas showing sequential coronal sections of the areas reached by injections within and outside the CeA. Fig. 2 is a photomicrograph of a transverse section of the forebrain of one rat, representative of the rats tested, showing the typical bilateral injection sites in the CeA. Injections reaching the medial, lateral and capsular portions of the CeA were observed in some rats and the results from these rats were included in the statistical analysis and the final number of rats used for statistical analysis was 71. Figs. 3 and 4 respectively display the 0.3 M NaCl and water intake obtained with

rats that received misplaced injections of DAMGO in rats submitted to the WD–PR and combinations of saline, DAMGO 2 nmol and CTAP 1 nmol in FURO + CAP-treated rats. No effect was observable when these drugs were injected into sites located outside CeA. Rats in which injections did not reach the CeA (misplaced injections) were excluded from the statistical analyses of data presented in Figs. 5–13. Effects of bilateral injections of DAMGO into the CeA on 0.3 M NaCl and water intake in rats submitted to the WD–PR Bilateral injections of DAMGO into the CeA in rats submitted to the WD–PR induced a dose-related

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Fig. 5. (A) Cumulative 0.3 M NaCl intake; (B) Cumulative water intake by rats submitted to the WD–PR that received bilateral injections of DAMGO in several doses or saline into the CeA (two-bottle test). ⁄ Denotes a statistically significant difference (P < 0.05) when the distinct treatment groups are compared to the WD–PR rats receiving CeA injections of saline. n = number of rats.

increase of 0.3 M NaCl intake and water intake (two-bottle test) (Fig. 5), as shown by the significant difference between treatments [0.3 M NaCl: F(3,27) = 7.488, P < 0.05; Water: F(3,27) = 3.591, P < 0.05] and the significant interaction between treatments and times [0.3 M NaCl: F(27,243) = 5.364, P < 0.05; Water: F(27,243) = 5.481, P < 0.05]. As expected, WD–PR rats receiving CeA injections of saline solution drink an amount of 0.3 M NaCl [F(1,9) = 7.552; P < 0.05] and water [F(1,9) = 25.936; P < 0.05] that is significantly greater than that presented by intra-CeA saline-treated rats not submitted to the WD–PR (no WD–PR) (Fig. 5). DAMGO (2 nmol) injected into CeA did not affect water intake in rats submitted to WD–PR when they had

access only to water during tests (one-bottle test) [F(1,8) = 0.003; P > 0.05] (Fig. 6). Effects of bilateral injections of combinations of DAMGO and CTAP into the CeA on 0.3 M NaCl and water intake in rats submitted to the WD–PR 0.5, 1, and 2 nmol doses of CTAP pretreatment all appeared to significantly suppress intra-CeA DAMGOinduced 0.3 M NaCl intake from 30 min until the end of the test (Fig. 7A) and water intake from 60 min until the end of the test (Fig. 7B) in a dose-dependent manner (two-bottle test). Two-way repeated measures ANOVA revealed significant main effects of drug treatment

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Fig. 6. Cumulative water intake by rats submitted to the WD–PR that received bilateral injections of DAMGO (2 nmol) or saline into the CeA when they had access only to water during tests (one-bottle test). n = number of rats.

[0.3 M NaCl: F(4,32) = 7.344, P < 0.05; water: F(4,32) = 11.649, P < 0.05], postinjection time [0.3 M NaCl: F(9,72) = 18.641, P < 0.05; water: F(9,72) = 56.303, P < 0.05], and their interaction [0.3 M NaCl: F(36,288) = 4.013, P < 0.05; water: F(36,288) = 9.902, P < 0.05].

Effects of bilateral injections of combinations of saline, DAMGO and CTAP into the CeA on FURO + CAP-induced 0.3 M NaCl and water intake As shown in Fig. 8, as expected, FURO + CAP-treated rats receiving CeA injections of saline solution drink an amount of 0.3 M NaCl [F(1,6) = 6.707; P < 0.05] and water [F(1,6) = 105.411; P < 0.05] that is significantly greater than that presented by saline injected into the CeA in rats treated with subcutaneous injections of isotonic saline solution instead of the diuretic FURO plus CAP (no FURO/CAP). Although CTAP (1 nmol) administration (CTAP + saline) into the CeA appeared to reduce 0.3 M NaCl intake, this trend did not reach statistical significance (Fig. 8A). In contrast, interestingly, CTAP at the same dose produced a statistically significant reduction of water intake that followed sodium intake (Fig. 8B) and a reduction of total fluid intake (0.3 M NaCl + water) (6.486 ± 0.548 ml/ 240 min vs. vehicle: 10.429 ± 1.683 ml/240 min) [F(1,6) = 6.332; P < 0.05]. However, CTAP (1 nmol) pretreatment statistically suppressed 0.3 M NaCl intake and water intake induced by DAMGO 2 nmol injected into the same area (two-bottle test) (Fig. 8). The main effect of drug treatment was significant [0.3 M NaCl: F(3,18) = 10.282, P < 0.05; water: F(3,18) = 23.206, P < 0.05], as was postinjection time [0.3 M NaCl: F(9,54) = 22.607, P < 0.05; water: F(9,54) = 24.742, P < 0.05] and their interaction [0.3 M NaCl:

F(27,162) = 6.400, P < 0.05; water: F(27,162) = 7.075, P < 0.05]. DAMGO (2 nmol) injected into CeA did not affect FURO + CAP-induced water intake when they had access only to water during tests (one-bottle test) [F(1,6) = 1.563; P > 0.05] (Fig. 9). Effects of bilateral injections of DAMGO into the CeA on water deprivation-induced water intake Fig. 10 exhibits the effects of CeA injections of DAMGO in different doses or isotonic saline solution on water intake in water-deprived rats. As expected, water-deprived rats receiving CeA injections of saline solution drink an amount of water [F(1,6) = 75.164; P < 0.05] that is significantly greater than that presented by intra-CeA saline-treated rats not submitted to water deprivation (no WD) (Fig. 10). Compared to the water-deprived rats receiving CeA injections of saline, all doses of DAMGO injected into the CeA in water-deprived rats produced no change in water intake [F(3,18) = 0.324; P > 0.05] (Fig. 10). Effects of bilateral injections of DAMGO into the CeA on renal excretion in rats As shown in Fig. 11, compared to saline injections, injections of DAMGO (2 nmol) into CeA produced no change in cumulative urinary volume [F(1,7) = 2.917; P > 0.05] and sodium excretion [F(1,7) = 0.046; P > 0.05]. Effects of bilateral injections of DAMGO into the CeA on arterial pressure in rats As shown in Fig. 12, compared to saline injections, injections of DAMGO (2 nmol) into CeA did not affect MAP [F(1,6) = 0.642; P > 0.05].

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Fig. 7. (A) Cumulative 0.3 M NaCl intake; (B) Cumulative water intake by rats submitted to the WD–PR that received bilateral injections of combinations of saline, DAMGO and CTAP into the CeA (two-bottle test). ⁄ Indicates a statistically significant difference (P < 0.05) when the distinct treatment groups are compared to the WD–PR rats receiving CeA injections of saline. # Denotes a statistically significant difference (P < 0.05) when the distinct treatment groups are compared to the WD–PR rats receiving CeA injections of DAMGO 2 nmol. n = number of rats.

Effects of bilateral injections of DAMGO into the CeA on activity in rats As shown in Fig. 13, compared to saline injections, injections of DAMGO (2 nmol) into CeA significantly increased horizontal activity [F(1,6) = 11.716; P < 0.05] (Fig. 13A) and vertical activity [F(1,6) = 11.853; P < 0.05] (Fig. 13B).

DISCUSSION These data strongly implicate l-ORs in the CeA in the excitatory modulation of sodium intake in rats. The selective l-OR agonist DAMGO has very high affinity

for l-ORs (0.14 nM) and displays at least 1000-fold selectivity in binding to these vs. j-and d-opioid recognition sites (Goldstein and Naidu, 1989). The highly selective l-OR antagonist CTAP is 10-fold more potent than naloxone (a nonselective opioid antagonist) at blocking l-ORs and has 1000-fold higher affinity for l-than d-ORs with poor affinity also for j-ORs (Kramer et al., 1989; Abbruscato et al., 1997; Heyliger et al., 1999). The present results show that bilateral injections of DAMGO into the CeA induce hypertonic NaCl ingestion and pretreatment with bilateral injections of CTAP into the CeA suppress the hypertonic NaCl intake induced by DAMGO injected into the same site in rats submitted to the WD–PR. The present results also show

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Fig. 8. (A) Cumulative 0.3 M NaCl intake; (B) Cumulative water intake by FURO + CAP-treated rats that received bilateral injections of combinations of saline, DAMGO 2 nmol and CTAP 1 nmol into the CeA (two-bottle test). ⁄ Indicates a statistically significant difference (P < 0.05) when the distinct treatment groups are compared to the FURO/CAP rats receiving CeA injections of saline. # Denotes a statistically significant difference (P < 0.05) when the distinct treatment groups are compared to the FURO/CAP rats receiving CeA injections of DAMGO 2 nmol. n = number of rats.

that bilateral injections of DAMGO into the CeA induce hypertonic NaCl ingestion and the blockade of l-ORs with CTAP injected into the CeA reduce the increase in 0.3 M NaCl intake induced by DAMGO injected into the same site in FURO + CAP-treated rats. Rats treated with DAMGO injected into the CeA also ingested water if hypertonic NaCl was simultaneously available (twobottle test), however, no water was ingested if rats had access only to water (one-bottle test), suggesting that the ingestion of water was a consequence of the increased plasma osmolarity due to the excessive

ingestion of hypertonic NaCl. Moreover, the present study shows that DAMGO injected into the CeA has no effect on water intake in water-deprived rats. In the present study, CTAP (1 nmol) administration into the CeA did not produce a statistical reduction of 0.3 M NaCl intake induced by FURO + CAP-treatment (Fig. 8A) but induced a statistically significant reduction of water intake that followed the sodium intake (Fig. 8B) and reduction of total fluid intake (0.3 M NaCl + water), suggesting the reduced extent of water consumption that followed sodium intake to adjust total fluid intake to

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Fig. 9. Cumulative water intake by FURO + CAP-treated rats that received bilateral injections of DAMGO (2 nmol) or saline into the CeA when they had access only to water during tests (one-bottle test). n = number of rats.

Fig. 10. Cumulative water intake by water-deprived rats treated with CeA injections of DAMGO in several doses or saline. ⁄ Denotes a statistically significant difference (P < 0.05) when the distinct treatment groups are compared to the water-deprived rats receiving CeA injections of saline. n = number of rats.

isotonic was more than the reduced extent of 0.3 M NaCl intake induced by 1 nmol CTAP administration into the CeA in FURO + CAP-treated rats. In addition, in the present study, injections of DAMGO into areas located outside the CeA did not produce any significant effects on 0.3 M NaCl and water intake in rats submitted to the WD–PR or in FURO + CAP-treated rats, indicating that the effects observed here are consequent to the pharmacological stimulation of l-ORs located within the CeA. Collectively, our results would appear to establish that activating the l-ORs in the CeA induces hypertonic sodium intake.

In a previous study, we found that DAMGO injected into the CeA also induced 0.3 M sucrose intake in water-deprived rats (Sun et al., 2012), which suggests that opioidergic mechanisms in the CeA are also involved in the control of sucrose intake. In the present study, DAMGO injected into the CeA induced no water intake in water-deprived rats. Therefore, the ingestion of hypertonic NaCl and sucrose solutions induced by DAMGO injected into the CeA is not due to non-specific activation of all ingestive behaviors. In addition, pharmacological activation of 5-HT3 receptors located within the CeA inhibited salt intake in sodium-depleted

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Fig. 11. (A) Cumulative urinary volume; (B) Cumulative urinary sodium excretion in rats treated with bilateral injections of DAMGO (2 nmol) or saline into the CeA. n = number of rats.

rats and failed to modify the ingestion of a palatable saccharin solution (Luz et al., 2007), which suggests that the increased ingestion of hypertonic NaCl by rats treated with DAMGO injections into the CeA in the present study is an effect more specific for DAMGO acting in the CeA. Sodium appetite may be strongly influenced by changes in blood pressure. Hypotension seems to stimulate whereas hypertension inhibits salt intake. Sodium intake is more promptly developed during hypovolemia if the animals are made simultaneously hypotensive (Johnson and Thunhorst, 1997). After the combined administration of furosemide plus captopril, a treatment that induces sodium depletion, salt intake is significantly reduced if blood pressure is not allowed to

decrease by the use of sympathomimetic drugs such as phenylephrine (Thunhorst and Johnson, 1994). In present study, the pharmacological stimulation of l-ORs located within the CeA was unable to modify arterial blood pressure and produced no change in renal excretion, suggesting that the natriorexigenic response to DAMGO injected into the CeA is not secondary to cardiovascular responses or increased renal excretion. Motor activity was elevated at the 15-min timepoint initially (Fig. 13), in contrast, DAMGO increased salt intake is seen later (Figs. 5 and 7). Similar observations have been reported for the increased ingestion of chow, hypertonic NaCl or sucrose solution despite elevated motor activity after the infusion of DAMGO into the lateral parabrachial nucleus (LPBN), striatum and the

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Fig. 12. Mean arterial pressure in rats treated with bilateral injections of DAMGO (2 nmol) or saline into the CeA. n = number of rats.

nucleus accumbens (Bakshi and Kelley, 1993; Zhang and Kelley, 1997; Wilson et al., 2003). So, we entertained the possibility that the hyperactivity and increase of salt intake induced by DAMGO injected into the CeA may be two independent events, and drawing a conclusion that the ingestion of hypertonic NaCl induced by DAMGO injected into the CeA is completely due to hyperactivity elicited by DAMGO injected into the same site seems arbitrary. The CeA is thought to receive integrated information from other amygdala subregions including the BLA which is believed to converge information on conditioned and unconditioned environmental stimuli and to modulate the process of memory consolidation (Pitkanen et al., 1997; McGaugh, 2002). The CeA produces its actions through extensive efferent projections to the basal forebrain, hypothalamus, midbrain, and brainstem nuclei that mediate fear response, reward behavior, and environmental analgesia (Pitkanen et al., 1997; Swanson and Petrovich, 1998; Davis, 2000). The CeA contains intrinsic neurons and axon terminals that contain opioid peptides (Fallon and Leslie, 1986; Cassell and Gray, 1989; Poulin et al., 2006) and l-ORs are present in the CeA (Mansour et al., 1995; Poulin et al., 2006; Glass et al., 2009). Most of neurons in the rat CeA are inhibited by l-OR agonists (Zhu and Pan, 2004; Chieng et al., 2006). This l-OR agonist-induced hyperpolarization was mediated by the opening of inwardly rectifying potassium channels (Zhu and Pan, 2004). Chieng et al. (2006) found no relationship between the location of CeA neurons and the postsynaptic expression of functional l-ORs, suggesting that a subset of projections from each major CeA subdivision is directly inhibited by the activation of l-ORs, given the anatomical evidence that CeA cells and their efferent projections are predominantly GABAergic (Swanson and Petrovich, 1998; Sah et al.,

2003; Zhu and Pan, 2004). Then, through l-ORs, endogenously released opioid peptides would inhibit those GABAergic projection neurons and reduce their inhibitory effect on the projection targets (Baxter and Murray, 2002; Zhu and Pan, 2004, 2005). We entertained the possibility that the reduced inhibition from the CeA caused by the activation of l-ORs within the CeA could contribute to facilitate the sodium intake. The CeA has reciprocal direct connections with the hindbrain, particularly the PBN and the nucleus of the solitary tract (NTS) (Norgren, 1995; Swanson and Petrovich, 1998). GABAergic connections between the CeA and the LPBN have been demonstrated and CeA is also directly connected with the NTS particularly with the region of the NTS rich in aldosterone-sensitive neurons that co-express the mineralocorticoid receptor and the enzyme 11-hydroxysteroid dehydrogenase type 2 (HSD2). The HSD2 neurons are activated by mineralocorticoids and deactivated by the ingestion of sodium (Jia et al., 2005; Geerling and Loewy, 2006, 2007). Visceral and gustatory informations that ascend to the NTS make a second relay in the PBN prior to project to the amygdala, thus forming a major neuroaxis for the control of taste and sodium appetite (Flynn et al., 1991; Johnson et al., 1999; Geerling and Loewy, 2006; Norgren et al., 2006). This particular pathway may be one of the neuroanatomical circuits explaining the modulation of salt intake by the CeA (Geerling and Loewy, 2006). Lately, our laboratory has also demonstrated that the microinjection of GABAA receptor agonist muscimol into the CeA not only inhibited sodium intake in sodium-depleted rats but also increased the number of Fos-like immunoreactive neurons in the NTS and LPBN, suggesting an important facilitatory mechanism related to the control of sodium appetite are present in the CeA and the CeA–PBN–NTS pathway is involved in mediating the sodium intake (Wang et al., 2012).

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Fig. 13. (A) Horizontal activity; (B) Vertical activity in rats treated with bilateral injections of DAMGO (2 nmol) or saline into the CeA. ⁄ Denotes P < 0.05 compared to the saline injections. n = number of rats.

Since lesions of the CeA (Galaverna et al., 1992; Zardetto-Smith et al., 1994) or activation of GABAA receptors in the CeA (Wang et al., 2012) decreases sodium intake, suggesting an important facilitatory mechanism for sodium intake in the CeA, the CeA is considered to be an essential area for sodium appetite (Fitzsimons, 1998; Geerling and Loewy, 2008). Important inhibitory mechanisms for the control of water and NaCl intake have been demonstrated in the LPBN (Edwards and Johnson, 1991; Callera et al., 2005). LPBN and CeA are strongly connected to control sodium appetite and it seems that the increase of sodium intake produced by the deactivation of LPBN inhibitory

mechanisms is totally dependent on facilitatory mechanisms present in the CeA (Andrade-Franze´ et al., 2010). Given the anatomical evidence that GABAergic connections exist between the CeA and the LPBN (Jia et al., 2005), it is possible that the disinhibition from the CeA caused by the activation of l-ORs within the CeA could reduce the CeA inhibitory effect on the LPBN, thus the LPBN inhibitory mechanisms are deactivated, and then hypertonic sodium intake is promoted. In addition, projections from the CeA have also been hypothesized to reduce the inhibitory activity of the hypothalamic paraventricular nucleus on the control of sodium appetite (Gray et al., 1989; Zardetto-Smith et al., 1994).

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CONCLUSION In conclusion, the results of the present study demonstrate that stimulating l-ORs in the CeA increases, whereas blocking those sites decreases, hypertonic sodium intake in rats submitted to the WD–PR and in FURO + CAP-treated rats. These pharmacological data, therefore, provide direct support for the CeA as a locus within the brain where opioids subserve the distributed neural network modulating sodium appetite.

AUTHOR CONTRIBUTIONS Junbao Yan, Jinrong Li and Jianqun Yan conception and design of research; Junbao Yan, Jinrong Li, Huiling Sun and Qian Wang performed experiments; Junbao Yan and Jianqun Yan analyzed the data; Junbao Yan, Jianqun Yan, Huiling Sun, Qian Wang, Jinrong Li, Ke Chen, Bo Sun, Xiaojing Wei and Lin Song interpreted the results of experiments; Junbao Yan drafted the manuscript; Junbao Yan and Jianqun Yan edited the manuscript; Junbao Yan, Jianqun Yan, Jinrong Li, Xiaolin Zhao, Shuangyu Wei and Ling Han revised the manuscript; Junbao Yan, Jinrong Li, Jianqun Yan, Huiling Sun, Qian Wang, Ke Chen, Bo Sun, Xiaojing Wei, Lin Song, Xiaolin Zhao, Shuangyu Wei and Ling Han approved the final version of the manuscript.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. Acknowledgment—The present work was supported by the National Natural Science Foundation of China (Nos. 31000518, 31171052 and 30970973).

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(Accepted 15 December 2012) (Available online 25 December 2012)