Facilitation of sodium intake by combining noradrenaline into the lateral parabrachial nucleus with prazosin peripherally

Pharmacology, Biochemistry and Behavior 111 (2013) 111–119

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Facilitation of sodium intake by combining noradrenaline into the lateral parabrachial nucleus with prazosin peripherally S. Gasparini a, J.M.C. Gomide a, G.M.F. Andrade-Franzé a, L.T. Totola b, L.A. De Luca Jr. a, D.S.A. Colombari a, P.M. De Paula a, T.S. Moreira b, J.V. Menani a,⁎ a b

Department of Physiology and Pathology, School of Dentistry, São Paulo State University, UNESP, Araraquara, São Paulo, Brazil Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo-USP, São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 22 March 2013 Received in revised form 20 August 2013 Accepted 28 August 2013 Available online 13 September 2013 Keywords: Noradrenaline Baro and volume receptors Parabrachial nucleus Sodium appetite Blood pressure

a b s t r a c t Injections of noradrenaline into the lateral parabrachial nucleus (LPBN) increase arterial pressure and 1.8% NaCl intake and decrease water intake in rats treated with the diuretic furosemide (FURO) combined with a low dose of the angiotensin converting enzyme inhibitor captopril (CAP). In the present study, we investigated the influence of the pressor response elicited by noradrenaline injected into the LPBN on FURO + CAP-induced water and 1.8% NaCl intake. Male Holtzman rats with bilateral stainless steel guide-cannulas implanted into LPBN were used. Bilateral injections of noradrenaline (40 nmol/0.2 μl) into the LPBN increased FURO + CAP-induced 1.8% NaCl intake (12.2 ± 3.5, vs., saline: 4.2 ± 0.8 ml/180 min), reduced water intake and strongly increased arterial pressure (50 ± 7, vs. saline: 1 ± 1 mm Hg). The blockade of the α1 adrenoceptors with the prazosin injected intraperitoneally abolished the pressor response and increased 1.8% NaCl and water intake in rats treated with FURO + CAP combined with noradrenaline injected into the LPBN. The deactivation of baro and perhaps volume receptors due to the cardiovascular effects of prazosin is a mechanism that may facilitate water and NaCl intake in rats treated with FURO + CAP combined with noradrenaline injected into the LPBN. Therefore, the activation of α2 adrenoceptors with noradrenaline injected into the LPBN, at least in dose tested, may not completely remove the inhibitory signals produced by the activation of the cardiovascular receptors, particularly the signals that result from the extra activation of these receptors with the increase of arterial pressure. © 2013 Elsevier Inc. All rights reserved.

1. Introduction The lateral parabrachial nucleus (LPBN) is a major site that receives ascending projections from the area postrema and the medial portion of the nucleus of the solitary tract (AP/mNTS), an important area of the hindbrain innervated by afferents from arterial baroreceptors, cardiopulmonary receptors, gustatory receptors and other visceral receptors that influence water and NaCl intake (Herbert et al., 1990; Lança and van der Kooy, 1985; Johnson, 2007; Johnson and Thunhorst, 1997, 2007; Norgren, 1981).

Abbreviations: ACE, angiotensin-converting enzyme; ANG II, angiotensin II; ANOVA, analysis of variance; AP, area postrema; CAP, captopril; DOI, 2,5-dimetoxy-4-iodoamphetamine hydrobromide; FURO, furosemide; HR, heart rate; 5-HT, serotonin; i.c.v., intracerebroventricularly; i.p., intraperitoneal; LPBN, lateral parabrachial nucleus; mNTS, medial portion of the nucleus of the solitary tract; NOR, noradrenaline; NTS, nucleus of the solitary tract; SAL, saline; s.c., subcutaneous; SCP, superior cerebellar peduncle; SFO, subfornical organ. ⁎ Corresponding author at: Department of Physiology and Pathology, School of Dentistry, Sao Paulo State University, UNESP, Rua Humaitá, 1680, Araraquara, 14801 903, SP, Brazil. Tel.: +55 16 3301 6486; fax: +55 16 3301 6488. E-mail address: [email protected] (J.V. Menani). 0091-3057/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2013.08.017

The blockade of serotonin, cholecystokinin (CCK), corticotrophin releasing factor (CRF) or glutamate receptors, or activation of α2-adrenoceptors in the LPBN increases hypertonic NaCl intake and occasionally also water intake produced by the treatment with the diuretic furosemide (FURO) combined with a low dose of the angiotensin-converting enzyme inhibitor captopril (CAP), both injected subcutaneously (s.c.), suggesting the existence of important inhibitory mechanisms for the control of sodium and water intake in the LPBN (Menani et al., 1996; Menani and Johnson, 1998; De Gobbi et al., 2001; Andrade et al., 2004; De Castro e Silva et al., 2006; De Gobbi et al., 2009; Gasparini et al., 2009). The inflation of a balloon placed at the superior vena cava-right atrial junction reduces 1.8% NaCl intake induced by FURO + CAP treatment and increases c-fos protein in the LPBN, suggesting that the LPBN might be a site that receives signals from cardiovascular receptors that inhibit sodium intake (De Gobbi et al., 2008). The changes in the activities of these cardiovascular receptors caused by reductions or increases in the arterial pressure might facilitate or inhibit, respectively, water and sodium intake (Thunhorst and Johnson, 1994; Johnson and Thunhorst, 1997, 2007; Johnson, 2007; De Gobbi et al., 2008). However, the injections of noradrenaline into the LPBN facilitate FURO + CAP-induced sodium intake, in spite of the

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strong increase of arterial pressure produced by these injections (Gasparini et al., 2009). The facilitation of FURO + CAP-induced 1.8% NaCl intake produced by the injections of noradrenaline into the LPBN similar to the facilitation produced by moxonidine (α2-adrenoceptor and imidazoline agonist) into the LPBN depends on the activation of the α2-adrenoceptors in the LPBN (Andrade et al., 2004; Gasparini et al., 2009). The only difference is that the activation of the α2-adrenoceptors with the injections of moxonidine into the LPBN does not modify arterial pressure (Andrade et al., 2004). This may explain why noradrenaline is less effective than moxonidine injected into the LPBN to increase FURO + CAP-induced sodium intake and also differently from moxonidine, noradrenaline reduces FURO + CAP-induced water intake. In the present study, we investigated the influence of the pressor response produced by noradrenaline on water and 1.8% NaCl intake by rats treated with s.c. FURO + CAP and the possible peripheral mechanisms (sympathetic activation and/or vasopressin secretion) that might be activated by the injections of noradrenaline injected into the LPBN to produce pressor responses. For this, we tested the effects of the blockade of the pressor response to noradrenaline injected into the LPBN with intraperitoneal (i.p.) injection of prazosin (specific α1 adrenoceptor antagonist) on FURO + CAP-induced water and 1.8% NaCl intake and the effects of the intravenous (i.v.) injection of a vasopressin antagonist combined with hexamethonium (ganglionic blocker) on the pressor and bradycardic responses to noradrenaline injected into the LPBN in unanesthetized rats. Additionally, we also evaluated the cardiovascular and sympathetic responses to noradrenaline injected into the LPBN in anesthetized rats.

2. Material and methods 2.1. Animals Male Holtzman rats (for tests in unanesthetized animals) or Wistar rats (for tests in anesthetized animals) weighing 280 to 320 g were used. The animals were housed individually in stainless steel cages in a room with controlled temperature (23 ± 2 °C) and humidity (55 ± 10%). The lights were on from 7:00 am to 7:00 pm. Guabi rat chow (Paulínia, SP, Brazil), tap water and 1.8% NaCl were available ad libitum. Animals' use was in accordance with the guidelines approved by the Animal Experimentation Ethics Committee of the Dentistry School of Araraquara — UNESP and Institute of Biomedical Science at the University of São Paulo (ICB/USP). All efforts were made to minimize animal discomfort and the number of animals used.

2.2. Brain surgery The animals were anesthetized with ketamine (80 mg/kg of body weight i.p.) and xylazine (7 mg/kg of body weight i.p.) and placed in a Kopf stereotaxic instrument. The skull was positioned to have the bregma and lambda at the same horizontal level. Stainless steel cannulas (12 × 0.6 mm o.d.) were implanted bilaterally in the LPBN (coordinates: 9.4 mm caudal to bregma, 2.1 mm lateral to midline and 4.1 mm below the dura mater). The tips of the cannulas were positioned at a point 2 mm above the LPBN. Besides the LPBN cannulas, one group of rats, received also stainless steel cannulas implanted into the lateral ventricle (LV, coordinates: 0.3 mm caudal to bregma, 1.5 mm lateral to midline and 3.5 mm below the dura mater). The cannulas were fixed to the cranium using dental acrylic resin and jeweler screws. At the end of the surgery, the animals received an intramuscular injection of penicillin (30,000 IU) and an s.c. injection of the analgesic Ketoflex (ketoprofen 1%, 0.03 ml/rat). After the surgery, rats were allowed to recover for one week before starting water and NaCl intake tests or arterial pressure recordings.

2.3. Arterial pressure and heart rate recordings in unanesthetized animals The mean arterial pressure and HR were recorded in unanesthetized rats. Under ketamine anesthesia (100 mg/kg of body weight i.p.) and xylazine (7 mg/kg of body weight i.p.), a polyethylene tubing (PE-10 connected to a PE-50) was inserted into the abdominal aorta through the femoral artery on the day before the experiments. At the same time, in some rats, a polyethylene tubing was also inserted in the femoral vein for drug administration. Both tubings were tunneled subcutaneously and exposed on the back of the rat to allow access in unrestrained, freely moving rats. To record pulsatile arterial pressure (PAP), MAP and HR, the arterial catheter was connected to a Statham Gold (P23 Db) pressure transducer connected with an ETH-200 amplifier (CB Sciences) and to a PowerLab data acquisition systems (ADInstruments).

2.4. Sympathetic nerve activity recording The rats were deeply anesthetized with halothane (5% in 100% oxygen in inspired air) for general surgical procedures, such as: a) tracheostomy for artificial ventilation; b) femoral artery and vein catheterization for arterial pressure measurement and administration of fluids and drugs, respectively; c) intracerebral injection by removal of the occipital bone and retracting the underlying dura mater membrane for insertion of a pipette into the medulla oblongata via a dorsal transcerebellar approach; d) splanchnic sympathetic nerve isolation for subsequent nerve activity monitoring. The level of anesthesia was checked by a flexor reflex to the animal's paw pinching. Splanchnic sympathetic nerve activity (sSNA) was recorded as previously described (Totola et al., 2013). Briefly, the right splanchnic nerve was isolated via a retroperitoneal approach, and the segment distal to the suprarenal ganglion was placed on a pair of Teflon-coated silver wires that had been bared at the tip (250 μm bare diameter; A-M Systems, www.a-msystems.com). The nerves and wires were embedded in adhesive material (Kwik-Cast Sealant, WPI, USP), and the wound was closed around the exiting recording wires. Upon completion of the surgical procedures, halothane was replaced by urethane (1.2 g/kg of body weight) slowly administered intravenously (i.v.). All rats were artificially ventilated with 100% oxygen throughout the experiment. The rectal temperature was maintained at 37 °C and the end tidal-CO2 was monitored throughout the experiment with a capnometer (CWE, Inc., Ardmore, PA, USA) that was calibrated twice per experiment against a calibrated CO2/N2 mix. The adequacy of the anesthesia was monitored during a 20 minute stabilization period by testing for the absence of withdrawal response and the lack of arterial pressure change to firm toe pinch. After these criteria were satisfied, the muscle relaxant pancuronium was administered at the initial dose of 1 mg/kg i.v. and the adequacy of anesthesia was thereafter gauged solely by the lack of increase in arterial pressure to firm toe pinch. Approximately hourly supplements of one-third of the initial dose of urethane were needed to satisfy these criteria during the course of the recording period (2–3 h). As previously described (Wenker et al., 2013; Totola et al., 2013), mean arterial pressure (MAP), sSNA and end-expiratory CO2 (etCO2) were digitized with a micro1401 (Cambridge Electronic Design), stored on a computer, and processed off-line with version 6 of Spike 2 software (Cambridge Electronic Design). Integrated splanchnic nerve activities (∫SNA) were obtained after the rectification and smoothing (τ = 2 s) of the original signal, which was acquired with a 30–300 Hz bandpass filter. ∫SNA was normalized within animals by assigning a value of 100 to resting SNA and a value of 0 to the minimum value recorded either during the administration of a dose of phenylephrine that saturated the baroreflex (5 μg/kg, i.v.) or after ganglionic blockade (hexamethonium; 10 mg/kg, i.v.).

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2.5. Drugs

2.10. Experimental protocols

All drugs were purchased from Sigma Chemical Co, St. Louis, MO, USA. Noradrenaline (norepinephrine bitartrate, 10, 15, 20 or 40 nmol/0.2 μl), was injected into the LPBN to test the effects on MAP and HR. Noradrenaline at the dose of 40 nmol/0.2 μl was used on ingestive behavior tests. Prazosin chloride (1 mg/kg of body weight) was injected i.p. Furosemide (10 mg/kg of body weight) and captopril (5 mg/kg of body weight) were injected s.c. [β-Mercapto-β-, β-cyclopentamethylenepropionyl, O-me-Tyr2, Arg8] vasopressin (Manning Compound, 10 mg/kg of body weight), arginin vasopressin (12.5 ng/rat) and hexamethonium chloride (30 mg/kg of body weight) were injected intravenously (i.v.). Noradrenaline, prazosin, vasopressin, Manning Compound and hexamethonium were dissolved in isotonic saline. The saline was injected into LPBN as control treatment. The doses of the drugs used were based on previous studies that injected these drugs centrally or peripherally (Gasparini et al., 2009; Menani et al., 2000; Menani and Johnson, 1998; Sugawara et al., 1999).

2.10.1. Water and 1.8% NaCl intake induced by FURO + CAP in rats treated with prazosin i.p. The rats with bilateral stainless steel guide-cannulas implanted into LPBN were treated with FURO (10 mg/kg b. wt.) combined with CAP (5 mg/kg b. wt.) s.c. Noradrenaline (40 nmol/0.2 μl) or saline was bilaterally injected into the LPBN 45 min after FURO + CAP-treatment. Prazosin (1 mg/kg of body weight) or saline was injected i.p. 15 min before the injections of noradrenaline or saline into the LPBN. The measurements of water and 1.8% NaCl intake started 15 min after the LPBN injections and were made at every 30 minutes during 180 min. To test the effects of noradrenaline into the LPBN combined with prazosin i.p., the rats treated with FURO + CAP received the following combination of treatments: saline i.p + saline into LPBN; saline i.p. + noradrenaline into LPBN; prazosin i.p. + saline into LPBN; prazosin i.p. + noradrenaline into LPBN. In each test, after the treatment with FURO + CAP, the group of rats was divided in two and half of the group received one of the treatments described above and the remaining animals received another treatment into the LPBN. The sequence of the treatments into LPBN and i.p. in each rat in the different tests was randomized and at the end of the experiments each rat received all the four treatments. The interval between two tests was at least 48 h.

2.6. Central injections The injections into the LPBN were made using 5 μl Hamilton syringes connected by a polyethylene tubing (PE-10) to injector cannulas (0.3 mm o.d.). The injector cannulas were 2 mm longer than the guide cannulas. The volume of injection into the LPBN was 0.2 μl in each site and into the LV 1 μl.

2.7. Water and 1.8% NaCl intake tests For the tests, water and 1.8% NaCl were provided from burettes with 0.1 ml divisions that were fitted with metal drinking spouts. Water and 1.8% NaCl intake was induced by s.c. injections of FURO (10 mg/kg of body weight) + CAP (5 mg/kg of body weight). Immediately after the treatment with FURO + CAP, the rats were maintained without water or 1.8% NaCl for 1 h. After this period, water and 1.8% NaCl were offered to the animals and the cumulative intake was recorded for 3 h. Saline or noradrenaline was injected into LPBN 15 min before the animals' access to water and 1.8% NaCl. When used, the α1-adrenoceptor antagonist, prazosin or saline was injected i.p. 15 min before the injections of the agonists. A recovery period of at least 2 days was allowed between tests. During the tests rats had no access to food.

2.8. Histology At the end of the experiments, the animals received bilateral injections of 2% Evans blue solution (0.2 μl) into LPBN. They were then deeply anesthetized with sodium thiopental (60 mg/kg of body weight) and perfused transcardially with 10% formalin. The brains were removed and fixed in 10% formalin for at least 2 days. Then, the brains were frozen, cut in 60 μm sections, stained with Giemsa and analyzed by light microscopy to confirm the central sites of the injections.

2.9. Statistical analysis The results were analyzed by two-way repeated measures ANOVA (counterbalanced design tests) or two-way ANOVA with treatments and times as factors or one way ANOVA. In all the cases, ANOVA was followed by Newman–Keuls test. Differences were considered significant at p b 0.05.

2.10.2. Cardiovascular responses to the injections of noradrenaline into the LPBN combined with prazosin i.p. in unanesthetized rats In order to confirm that prazosin (1 mg/kg of body weight) i.p. was also able to block the pressor responses to the same dose of noradrenaline (40 nmol/0.2 μl) injected bilaterally into the LPBN in the behavior tests, the rats treated with FURO (10 mg/kg b. wt.) combined with CAP (5 mg/kg b. wt.) s.c. received bilateral injections of saline and noradrenaline (40 nmol/0.2 μl) into the LPBN 40 and 45 min, respectively, after FURO + CAP-treatment. Prazosin (1 mg/kg of body weight) or saline was injected i.p. 15 min before the injections of noradrenaline into the LPBN. MAP and HR were recorded for an additional 30 min period after the injections of noradrenaline. MAP and HR were recorded in the same group of rats in two different sessions with a 24 h interval between them. In one session the rats received an injection of prazosin i.p. and in the other saline i.p. The order of the injection of saline or prazosin i.p. in each session in different rats was randomized. 2.10.3. Cardiovascular responses to noradrenaline injected into the LPBN in unanesthetized rats that received hexamethonium combined with vasopressin receptor antagonist injected i.v. In order to investigate the possible mechanisms involved in the pressor response to noradrenaline injected into the LPBN we tested the effects of the ganglionic blockade with i.v. hexamethonium combined with the blockade of vasopressin receptors with i.v. injection of Manning Compound. Normotensive fluid replete unanesthetized rats with stainless steel guide-cannulas implanted in the LV and bilaterally in the LPBN and with polyethylene tubing inserted into the abdominal aorta through the femoral artery and into the femoral vein were used. After a period of control recording of baseline MAP and HR (around 20 min), rats received i.v. injections of vasopressin antagonist (Manning Compound, 10 μg/kg of body weight) or saline combined with i.v. injections of hexamethonium (30 mg/kg of body weight) or saline. Ten and 15 min later, the animals received unilateral injection of saline and noradrenaline (20 nmol/0.2 μl) into LPBN, respectively. Fifteen minutes after the injection of noradrenaline into the LPBN, the rats received i.c.v. injection of carbachol (4 nmol/1 μl). MAP and HR were recorded for an additional 15 min period after the injection of carbachol. MAP and HR were recorded in two different sessions with a 24 h interval between them. In one session the rats received injection of saline i.v. and in the other the combination of vasopressin antagonist and hexamethonium i.v.

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before noradrenaline. The order of the i.v. treatments in each session in different rats was randomized. Before the i.v. injections of the vasopressin antagonist combined with hexamethonium and after the injections of noradrenaline into the LPBN, rats received i.v. injections of vasopressin (12.5 ng/rat) to confirm the vasopressinergic receptor blockade. 2.10.4. Cardiovascular and sympathetic responses produced by injection of noradrenaline into the LPBN in anesthetized rats These experiments were performed in rats anesthetized with urethane (1.2 g/kg, i.v.). The recordings began 10 min after the connection of the arterial line to the pressure transducer. The mean arterial pressure (MAP) and splanchnic sympathetic nerve activity (sSNA) were continuously recorded for 70–80 min. The control (baseline) values were recorded for 10–20 min and were analyzed immediately before the vehicle injection (first treatment). These values were used as a reference to calculate the changes produced by the treatments. After 10 min, noradrenaline (20 nmol/0.2 μl) was injected unilaterally into the LPBN and the MAP and sSNA responses were evaluated immediately. The same experimental protocol was repeated twice during the experiment. 3. Results 3.1. Histological analysis The LPBN injection sites were centered in the central lateral or dorsal lateral portions of the LPBN (see Fulwiler and Saper, 1984, for definitions of the LPBN subnuclei) (Fig. 1). The sites of the injections into the LPBN in the present study were similar to those of previous studies showing the effects of the injections of methysergide, moxonidine or noradrenaline into the LPBN on NaCl and water intake (Menani et al., 1996; Menani and Johnson, 1998; De Gobbi et al., 2001; Andrade et al., 2004; De Gobbi et al., 2009; Gasparini et al., 2009). The results from rats with injections outside the LPBN were also analyzed. Fig. 1B to G illustrates the most common sites of injections outside the LPBN. 3.2. Water and 1.8% NaCl intake induced by FURO + CAP in rats treated with noradrenaline into LPBN combined with prazosin i.p. Bilateral injections of noradrenaline (40 nmol/0.2 μl) into the LPBN increased FURO + CAP-induced 1.8% NaCl intake [F (3, 30) = 5.89; p b 0.05] (Fig. 2A) and reduced water intake in the first 90 min of the test [F (3, 30) = 5.69; p b 0.05] (Fig. 2B). The injections of noradrenaline into the LPBN combined with prazosin (1 mg/kg of body weight) i.p. further increased FURO + CAP-induced 1.8% NaCl intake (Fig. 2A) and, in addition, also increased water intake (Fig. 2B). The injection of prazosin i.p. alone slightly increased water intake in fluid replete rats or in rats treated with FURO + CAP, without changing 1.8% NaCl intake (Fig. 2, Table 1). Prazosin i.p. combined with noradrenaline injected into LPBN produced no change of 1.8% NaCl or water intake by fluid replete rats (Table 1). 3.3. Cardiovascular responses to the injections of noradrenaline into the LPBN combined with prazosin i.p. in unanesthetized rats The bilateral injections of noradrenaline (40 nmol/0.2 μl) into the LPBN in rats treated with FURO + CAP s.c. increased MAP [F (3, 15) = 38.53; p b 0.05] and reduced HR [F (3, 15) = 9.38; p b 0.05] (Fig. 3, Table 2). The i.p. injection of prazosin (1 mg/kg of body weight) abolished the pressor and bradycardic responses to bilateral injections of noradrenaline (40 nmol/0.2 μl) injected into the LPBN in rats treated with FURO + CAP s.c. (Fig. 3, Table 2). The i.p. injections of prazosin in FURO + CAP-treated rats produced a long lasting reduction in MAP that reached the maximum of −34 ±

3 mm Hg compared to saline i.p. (2 ± 3 mm Hg) [F (2, 19) = 58.14; p b 0.05] and increased HR (101 ± 10 bpm, vs. saline i.p.: 20 ± 12 bpm) [F (2, 19) = 4.02; p b 0.05] (Fig. 3). 3.4. Cardiovascular responses to noradrenaline injected into the LPBN in unanesthetized rats that received hexamethonium combined with vasopressin receptor antagonist injected i.v. To investigate the possible mechanisms involved in the pressor response to noradrenaline injected into the LPBN we tested the effects of the ganglionic blockade with i.v. hexamethonium combined with the blockade of vasopressin receptors with i.v. injection of Manning Compound. The pressor response to noradrenaline (20 nmol/0.2 μl) injected unilaterally into LPBN increased in rats pre-treated with the vasopressinergic receptor antagonist (Manning Compound, 10 μg/kg of body weight) combined with hexamethonium (30 mg/kg of body weight) i.v. [F (3, 12) = 15.83; p b 0.05] (Fig. 4A). However, the bradycardic response to noradrenaline injected into the LPBN was abolished with this pre-treatment [F (3, 12) = 3.66; p b 0.05] (Fig. 4B). In the same animals, the vasopressinergic antagonist combined with hexamethonium i.v. abolished the pressor response to carbachol (4 nmol/1 μl) injected i.c.v. (Fig. 4A), confirming the efficiency of the combined sympathetic and vasopressinergic blockade. The pressor response to i.v. vasopressin (12.5 ng/0.1 ml/rat) was abolished by the i.v. injection of the vasopressinergic receptor antagonist (2 ± 4 mm Hg, vs. control pre-antagonist: 36 ± 3 mm Hg) which also confirms the efficiency of the blockade. 3.5. Cardiovascular and sympathetic responses produced by injection of noradrenaline into the LPBN in anesthetized rats In urethane-anesthetized rats (basal MAP: 122 ± 6 mm Hg, n = 5), the unilateral injection of noradrenaline (20 nmol/0.2 μl) into the LPBN increased MAP (30 ± 5, vs. saline: 4 ± 3 mm Hg, p b 0.05) and decreased sSNA (−22 ± 8%, vs. saline: 4 ± 2%, p b 0.05). The injection of the same dose of noradrenaline (20 nmol) intravenously also increased MAP (44 ± 10, vs. saline: 1 ± 1 mm Hg, p b 0.01) and reduced sSNA (−83 ± 2%, vs. saline: 1 ± 2%, p b 0.01). 3.6. Water and 1.8% NaCl intake induced by FURO + CAP and cardiovascular responses to noradrenaline injected outside the LPBN To test the specificity of the LPBN as the site in which the injections of noradrenaline produced effects on water and 1.8% NaCl intake and arterial pressure, the responses noradrenaline injected outside the LPBN were also analyzed. Bilateral injections of noradrenaline (40 nmol/0.2 μl) in sites outside the LPBN in unanesthetized rats also increased MAP (39 ± 4) and decreased HR (−82 ± 15) similarly to the injection into the LPBN (Table 3). Injections of noradrenaline (40 nmol/0.2 μl) in sites outside the LPBN did not modify FURO + CAP-induced 1.8% NaCl intake (Table 4). 4. Discussion A previous study demonstrated that noradrenaline acting on α2 adrenoceptors in the LPBN facilitates sodium intake induced by FURO + CAP treatment (Gasparini et al., 2009). The present results confirm this effect of noradrenaline injected into the LPBN and show that the blockade of the α1 adrenoceptors with prazosin injected i.p. abolished the pressor response to noradrenaline and increased 1.8% NaCl compared to the intake after combining s.c. FURO + CAP with noradrenaline into the LPBN. Water intake induced by FURO + CAP was reduced by noradrenaline injected into the LPBN and increased by combining noradrenaline into the LPBN with prazosin i.p. These results

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suggest that the deactivation of baroreceptors and perhaps volume receptors due to the cardiovascular effects of prazosin is a possible mechanism that facilitates water intake and increases the facilitation of NaCl intake in rats treated with FURO + CAP combined with noradrenaline injected into the LPBN. Although prazosin i.p. abolished the pressor response to noradrenaline injected into the LPBN, the ganglionic blockade with hexamethonium combined with the blockade of peripheral vasopressin receptors increased, rather than decreased, the pressor response to noradrenaline

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injected into the LPBN. Additionally, experiments performed in anesthetized rats showed that an injection of noradrenaline into the LPBN produced a pressor response and a decrease in sympathetic outflow, an effect opposite to that expected for a central acting hypertensive agent. Therefore, the results strongly suggest that the pressor response results from noradrenaline acting peripherally and not centrally. Afferent signals from arterial baroreceptors and cardiopulmonary receptors that reach the AP/mNTS ascend to the LPBN and can inhibit the ingestion of water and sodium (Johnson, 2007; Johnson and

Fig. 1. Photomicrographs of coronal sections of rat brains showing (A) the injection sites into the LPBN and (B, C, D, E, F and G) the injections in sites outside the LPBN (arrows). scp, superior cerebellar peduncle.

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SAL i.p. + SAL LPBN SAL i.p. + NOR LPBN PRAZOSIN i.p. + NOR LPBN PRAZOSIN i.p. + SAL LPBN * different from SAL i.p. + SAL LPBN # different from SAL i.p. + NOR LPBN

A Cumulative 1.8% NaCl intake (ml)

Thunhorst, 1997, 2007; Norgren, 1981). In addition, the LPBN may receive other visceral signals important to the control of sodium and water intake, like signals from gustatory receptors or osmoreceptors (Franchini and Vivas, 1999; Kobashi et al., 1993; Norgren, 1995; Yamamoto et al. 1993). The inflation of a balloon placed at the superior vena cava-right atrial junction reduces 1.8% NaCl intake produced by FURO + CAP treatment and increases c-fos protein in the LPBN (De Gobbi et al., 2008). The activation of the superior vena cava-right atrial junction receptors also attenuates isoproterenol-induced water intake in intact rats, and this inhibition is abolished by electrolytic lesions of the LPBN (Ohman and Johnson, 1995). These results suggest that the LPBN is a site that receives signals from cardiovascular receptors that inhibit sodium and water intake. The present results show that the pre-treatment with prazosin i.p. abolished the pressor response and increased water and NaCl intake by rats treated with FURO + CAP combined with noradrenaline injected into the LPBN. In addition to the blockade of the pressor response to noradrenaline, prazosin i.p. also reduced baseline arterial pressure and this might also reduce the inhibition produced by cardiovascular receptor activity. Therefore, by blocking the pressor response to noradrenaline injected into the LPBN and/or reducing the baseline arterial pressure, prazosin injected i.p. causes the unloading of baroreceptors and possible volume receptors which might facilitate water and NaCl intake. In this case, it seems that inhibitory signals from cardiovascular receptors (those removed by prazosin i.p.) still affect water and NaCl intake after the injections of noradrenaline into the LPBN, which suggests that the activation of α2 adrenoceptors with noradrenaline injected into the LPBN, at least in the dose tested, may not completely remove the inhibitory signals produced by the activation of the cardiovascular receptors, particularly the signals that result from the extra activation of these receptors produced by the increase of arterial pressure. Studies that have investigated the ingestion of 1.8% NaCl and water by FURO + CAP-treated rats suggest that the small reduction of the arterial pressure (around 10 mm Hg) produced by this treatment is important to stimulate the intake (Thunhorst and Johnson, 1994). Although the peak of the pressor response to noradrenaline was transitory and occurred before the intake, a long lasting small increase of arterial pressure is maintained for at least 30 min (Δ = 12 ± 4 mm Hg at 30 min after noradrenaline injection) which is the opposite to the effects of FURO + CAP-treatment, and, therefore, might inhibit sodium and water intake. The suggestion that the blockade of inhibitory signals by α2 adrenoceptor activation in LPBN facilitates FURO + CAP-induced hypertonic NaCl intake is reinforced by the results of a previous study that demonstrated that the activation of α2 adrenoceptors with the injections of moxonidine (an α2 adrenoceptors/imidazoline receptor agonist) into the LPBN reduces the aversive responses, and increases the ingestive responses, to an intra-oral infusion of hypertonic NaCl in rats (Andrade et al., 2011). A main difference between the injection of noradrenaline and moxonidine into the LPBN is the absence of pressor response when moxonidine is injected into the LPBN (Andrade et al., 2004). The activation of LPBN α2 adrenoceptors by moxonidine injection without changes in arterial pressure produces an intense facilitation of sodium intake probably as a consequence of the reduced activation of inhibitory signals in this condition, differently from the injection of noradrenaline into the LPBN that produces an extra activation of the inhibitory mechanisms due to the pressor responses. Moxonidine injected into the LPBN in a small dose (0.5 nmol/0.2 μl) strongly enhances FURO + CAP-induced 0.3 M NaCl intake (the mean ingestion of rats reaches 40 ml in 2 h) (Andrade et al., 2004). Although noradrenaline also activates α2 adrenoceptors in the LPBN, the ingestion of 0.3 M NaCl by rats treated with FURO + CAP combined with noradrenaline injected into the LPBN is reduced compared to that of rats treated with moxonidine into the LPBN. Rats treated with noradrenaline into the LPBN only reach an ingestion of 0.3 M NaCl similar to that of rats treated with moxonidine into the LPBN when they were previously treated with prazosin i.p., which again reinforces the

20

#

#

*

*

#

#

*

*

*

*

150

180

n = 11 15

10

5

0 0

30

60

90

120

Time (min)

B 20

Cumulative water intake (ml)

116

#

*

#*

#

# 15

# #

#

#

#

10

*

* 5

* 0 0

30

60

90

120

150

180

Time (min) Fig. 2. Cumulative (A) 1.8% NaCl intake and (B) water intake induced by FURO + CAP in rats treated with bilateral injections of noradrenaline (NOR, 40 nmol/0.2 μl) or saline (SAL) into the LPBN combined with prazosin (1 mg/kg of body weight) or SAL i.p. The results are reported as means ± SEM. n = number of animals.

suggestion that the inhibitory signals probably from cardiovascular origin are those that reduce the facilitation of sodium intake produced noradrenaline injected into the LPBN. In addition to cardiovascular signals, the LPBN also receives other inhibitory signals of sodium ingestion, like osmoreceptor signals, that might be modified by the activation of α2 adrenoceptors in this area facilitating sodium intake (Andrade et al., 2011). Moreover, besides α2 adrenergic mechanisms, other different

Table 1 Cumulative 1.8% NaCl and water intake by fluid replete rats treated with bilateral injections of noradrenaline or saline into LPBN combined with prazosin or saline i.p. Treatments

1.8% NaCl intake (ml/2 h)

Water intake (ml/2 h)

SAL i.p. + SAL LPBN SAL i.p. + NOR LPBN Prazosin i.p. + SAL LPBN Prazosin i.p. + NOR LPBN

0.6 0.3 0.7 1.5

0.9 0.3 2.8 2.2

± ± ± ±

0.3 0.2 0.3 0.9

± ± ± ±

0.6 0.2 0.6a 0.7

The results are reported as means ± SEM. Number of animals = 6. Noradrenaline (NOR, 40 nmol/0.2 μl); prazosin (1 mg/kg of body weight); saline (SAL). a Different from SAL + SAL.

S. Gasparini et al. / Pharmacology, Biochemistry and Behavior 111 (2013) 111–119

117

SAL noradrenaline (LPBN) (LPBN)

A

Time (min)

prazosin (i.p.)

SAL (LPBN)

noradrenaline (LPBN)

B

Time (min) Fig. 3. Representative recordings showing the changes in PAP, MAP and HR in one rat treated with FURO + CAP s.c. that received bilateral injections of saline (SAL) or noradrenaline (NOR, 40 nmol/0.2 ml) into LPBN combined with prazosin (1 mg/kg of body weight) i.p.

neurotransmitters and receptors like serotonin, CCK, CRF, glutamate, opioids, ATP and GABA in the LPBN modulate the inhibitory mechanisms that control sodium intake (Menani et al., 1996; Menani and Johnson, 1998; De Gobbi et al., 2001; Andrade et al., 2004; Callera et al., 2005; De Castro e Silva et al., 2006; De Oliveira et al., 2008; De Gobbi et al., 2009; Gasparini et al., 2009; Menezes et al., 2011). Therefore, the signals not modified or only partially modified by α2 adrenoceptor activation in the LPBN, like the cardiovascular signals as suggested by the present results, might be modulated or mediated by other neurotransmitters in the LPBN. Which neurotransmitters/ receptors mediate or modulate each signal is an important step for future studies. Although the activation of α2 adrenoceptors with injections of noradrenaline specifically into the LPBN is the mechanism that facilitates FURO + CAP-induced 1.8% NaCl intake (Gasparini et al., 2009 and the present results), the central site and the mechanisms involved in the pressor response are not clear. The injections of the same dose of

noradrenaline into the LPBN or outside the LPBN produced similar pressor and bradycardic responses, suggesting that the pressor response is not dependent specifically on the action of noradrenaline into the

Table 2 Changes in MAP and HR produced by bilateral injections of noradrenaline (NOR, 40 nmol/ 0.2 μl) or saline into LPBN combined with prazosin or saline i.p. in rats treated with FURO + CAP. Treatments

Δ MAP (mm Hg)

ΔHR (bpm)

SAL i.p. + SAL LPBN SAL i.p. + NOR LPBN Prazosin i.p. + NOR LPBN Prazosin i.p. + SAL LPBN

1 50 9 0.7

1 −62 −13 0.4

± ± ± ±

1 7a 4 0.8

± ± ± ±

4 13a 13 4

The results are reported as means ± SEM. Number of animals = 6. Prazosin (1 mg/kg of body weight.), noradrenaline (NOR, 40 nmol/0.2 μl), saline (SAL). a Different from SAL + SAL.

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S. Gasparini et al. / Pharmacology, Biochemistry and Behavior 111 (2013) 111–119

SAL + NOR HEXA + AVPx + NOR SAL + CARBACHOL HEXA + AVPx + CARBACHOL

Table 4 Cumulative 1.8% NaCl in FURO + CAP rats treated with bilateral injections of noradrenaline or saline outside LPBN combined with prazosin or saline i.p.

* different from SAL + NOR # different from SAL + carbachol

A

*

100

Δ MAP (mmHg)

80

*

1.8% NaCl intake (ml/3 h)

SAL i.p. + SAL LPBN SAL i.p. + NOR LPBN Prazosin i.p. + NOR LPBN Prazosin i.p. + SAL LPBN

6.5 7.8 6.3 3.9

± ± ± ±

2.3 1.4 2.2 1.4

The results are reported as means ± SEM. Number of animals = 6. Prazosin (1 mg/kg of body weight), noradrenaline (NOR, 40 nmol/0.2 μl), saline (SAL).

n=5

60 40 20 0

#

#

-20

#

#

# -40 0

1

2

3

4

5

Time (min)

B 100 75 50

Δ HR (bpm)

Treatment

25

*

0 -25

*

-50 -75

-100 -125 -150 0

1

2

3

4

5

Time (min) Fig. 4. Changes in (A) MAP and (B) HR produced by unilateral injections of noradrenaline (NOR, 20 nmol/0.2 μl) into the LPBN or carbachol (4 nmol/1 μl) i.c.v. In fluid replete rats pre-treated with injections of saline (SAL) or hexamethonium (HEXA, 30 mg/kg of body weight) + vasopressin antagonist (AVPx, 10 μg/kg of body weight) injected i.v. The results are reported as means ± SEM. n = number of animals.

LPBN. Sympathetic activation and/or vasopressin secretion are the mechanisms usually involved in the pressor responses produced by the stimulation of central areas, like the pressor responses produced by noradrenaline injected into the bed nucleus of the stria terminalis, supra-optic nucleus, lateral septal area or insular cortex or the cholinergic activation of forebrain areas (Alves et al., 2011; Busnardo et al., 2009; Crestani et al., 2007, 2008; Hoffman et al., 1977; Imai et al., 1989;

Table 3 Changes in MAP and HR produced by bilateral injections of noradrenaline or saline outside or into LPBN. Treatments

Δ MAP (mm Hg)

ΔHR (bpm)

Saline NOR LPBN NOR outside LPBN

3±3 41 ± 7a 39 ± 4a

6±3 −81 ± 23a −82 ± 15a

The results are reported as means ± SEM. Number of animals = 6. Noradrenaline (NOR, 40 nmol/0.2 μl). a Different from saline.

Menani et al., 1990; Pelosi and Corrêa, 2005; Scopinho et al., 2006; Takakura et al., 2011). Although, as expected, the double blockade (vasopressinergic receptor and ganglionic blockade) abolished the pressor response to central cholinergic activation, it increased the pressor responses to injections of noradrenaline into the LPBN, rather than reducing them, suggesting that noradrenaline injected into the LPBN does not activate mechanisms usually involved in the pressor responses that result from the activation of the central areas or that noradrenaline is not acting centrally to produce pressor responses. However, the double blockade abolished the bradycardic response to noradrenaline injected into the LPBN and the pressor response to intravenous injection of vasopressin, confirming that hexamethonium treatment produced an effective ganglionic blockade (in this case also the parasympathetic ganglion involved in the bradycardia) and that the vasopressin antagonist effectively blocked peripheral vasopressin receptors. The absence of bradycardia after the double blockade might be the reason for the increased pressor response to noradrenaline in this condition. Although hexamethonium combined with vasopressin antagonist i.v. did not reduce the pressor response to noradrenaline, prazosin i.p. abolished the pressor responses to noradrenaline injected into the LPBN which suggests that noradrenaline injected into the LPBN may spread to the periphery and activate α1 adrenoceptors located in the blood vessels to produce pressor response. The experiments performed in anesthetized rats also showed that the injection of noradrenaline into the LPBN produces pressor response and an inhibition of sympathetic outflow, an effect opposite to that expected for a central acting hypertensive agent. Therefore, the results show that noradrenaline injected into the LPBN does not increase sympathetic activity and, instead of this, strongly suggest that noradrenaline is probably diffusing outside the brain to produce vasoconstriction acting directly on α1 adrenoceptors in the blood vessels. In summary, the present results suggest that inhibitory signals from cardiovascular receptors still affect water and NaCl intake after the injections of noradrenaline into the LPBN. Therefore, it seems that the activation of α2 adrenoceptors with noradrenaline injected into the LPBN, at least in dose tested, may not completely remove the inhibitory signals produced by the activation of the cardiovascular receptors, particularly the signals that result from the extra activation of these receptors produced by the increase of arterial pressure. In addition, the pressor responses that result from the injection of noradrenaline into the LPBN seem to depend on a direct activation of peripheral α1 adrenoceptors and not on a central action of noradrenaline.

Acknowledgments The authors thank Silas P. Barbosa, Reginaldo C. Queiroz and Silvia Fóglia for their expert technical assistance, Silvana A. D. Malavolta for her secretarial assistance and Ana V. de Oliveira and Adriano Palomino for the animal care. This research was supported by public funding from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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