Inhibition of salt appetite in sodium-depleted rats by carvacrol: Involvement of noradrenergic and serotonergic pathways

European Journal of Pharmacology 854 (2019) 119–127

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Inhibition of salt appetite in sodium-depleted rats by carvacrol: Involvement of noradrenergic and serotonergic pathways

T

Filip de Souza Pollia,∗, Jefferson Novaes Gomesa, Hilda Silva Ferreirab, Rejane Conceição Santanaa, Josmara Bartolomei Fregonezea a b

Department of Physiology, Health Sciences Institute, Federal University of Bahia, 40110-100, Salvador, Bahia, Brazil Life Sciences Department, Bahia State University, 41195-001, Salvador, Bahia, Brazil

ARTICLE INFO

ABSTRACT

Keywords: Carvacrol Sodium appetite Serotonin Noradrenaline c-FOS

Carvacrol, a monoterpene phenol present in the essential oil of oregano, possesses several biological properties, such as antioxidant, anti-inflammatory, anxiolytic, anticonvulsive and antinociceptive. In vitro studies have shown that carvacrol inhibits serotonin, noradrenaline and dopamine transporters and the enzymes monoamine oxidase-A and B. Different brain functions are controlled by monoamines, including cardiovascular control, thirst and sodium appetite. In the present study we investigated the effects of intracerebroventricular (i.c.v.) injection of carvacrol on sodium appetite, and the participation of brain serotonergic and noradrenergic pathways on carvacrol effects. Neuronal activation in homeostasis-related brain areas induced by i.c.v. injection of carvacrol was also evaluated. Carvacrol dose-dependently inhibited hypertonic saline intake (1.5%) in sodiumdepleted rats, and this antinatriorexigenic effect was reduced by brain serotonergic depletion and by alphaadrenergic blockade. Furthermore, i.c.v. injections of carvacrol significantly increased the neuronal activation in brain areas involved in the control of salt appetite, such as MnPO, OVLT, PVN, SON, CeA and MeA. Taken together, our data show that carvacrol presents antinatriorexigenic activity through serotonin and noradrenaline pathways within brain circuits involved in the modulation of the body fluid homeostasis.

1. Introduction The use of plants for therapeutic purposes has been increasing in the last decades. The pharmacological properties of plants extracts are determined by the chemical components present among the secondary metabolites produced by the plant as a natural defense mechanism, mainly against herbivory. These secondary metabolites can be found in specific plant essential oils which are commercially available (Perricone et al., 2015), and might include aldehydes and phenols, such as carvacrol, citral, eugenol, thymol, and cinnamaldehyde (Bassolé and Juliani, 2012; Paduch et al., 2007). The main component of the essential oil of oregano (Origanum sp.) is carvacrol (CVC), a monoterpene phenol which can be present to as high as 80% of certain Origanum taxa essential oil (Baser, 2008; Verma et al., 2010). It has been shown that carvacrol presents several biological properties such as antibacterial, antifungal, antiprotozoal, antitumor and anticarcinogenic (Baser, 2008; Tepe et al., 2016), as well as exerts antioxidant and anti-inflammatory effects (Guimarães et al., 2012, 2010). Dose-dependent effects of carvacrol has been reported in reducing cancer cell number, fungi activity, blood pressure and nociception (Koparal and Zeytinoglu, 2003; ∗

Bayramoglu et al., 2006; Guimarães et al., 2010; Dantas et al., 2015). Moreover, carvacrol also presents actions in the central nervous system, displaying anxiolytic-like, anticonvulsive and antinociceptive effects, as well as cognition improvement in rat models of diabetis and dementia (Azizi et al., 2012; Deng et al., 2013; Kessler et al., 2014; Melo et al., 2010). It has been demonstrated that carvacrol inhibits serotonin, noradrenaline and dopamine transporters and the enzymes monoamine oxidase-A and B in vitro (Mechan et al., 2011), however, carvacrol actions in the central nervous system remains largely unexplored. The brain monoamine signaling is involved in several functions including cognition, mood, cardiovascular control, thirst and sodium appetite. Many studies have shown that the brain serotonergic pathways have an inhibitory or stimulatory effect on sodium appetite depending on the brain area and the type of serotonin receptor activated (De Luca et al., 2003; Franchini et al., 2002; Fregoneze et al., 2014; Hurley and Johnson, 2015; Johnson, 2007; Johnson and Thunhorst, 1997; Vivas et al., 2014). Noradrenaline is also involved in the regulation of the sodium appetite (De-Luca and Menani, 1997; De Paula et al., 1996; Sato et al., 1996; Sugawara et al., 1999; Yada et al., 1997). Since carvacrol has been shown to alter monoamine reuptake

Corresponding author. University of Copenhagen, Department of Drug Design and Pharmacology, Jagtvej 160, 2100, Copenhagen, Denmark. E-mail address: [email protected] (F.d.S. Polli).

https://doi.org/10.1016/j.ejphar.2019.04.026 Received 25 October 2018; Received in revised form 29 March 2019; Accepted 11 April 2019 Available online 13 April 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

European Journal of Pharmacology 854 (2019) 119–127

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complexes, affecting monoamine signaling, which is important in the regulation of sodium appetite, in the present study we investigated the effects of acute intracerebroventricular (i.c.v.) injection of carvacrol on sodium appetite and the participation of brain serotonergic and noradrenergic pathways in the responses induced by this phytochemical monoterpene. It was also evaluated whether i.c.v. injection of carvacrol was able to promote neuronal activation in brain areas involved in the control of sodium appetite through measurements of Fos expression, a nuclear protein expressed by the early gene c-fos, associated with neuronal activation upon a wide range of stimuli.

Both prazosin and pCPA were dissolved in isotonic saline and the control group received 5 μl of isotonic saline. Furosemide (Sanofi-Aventis Famacêutica Ltda, São Paulo, Brazil) was injected subcutaneously at the dose of 20 mg/kg. 2.4. Sodium depletion and hypertonic saline solution intake

One-hundred seventy one male Wistar rats (250–280 g) were kept under controlled light (lights on from 6 a.m. to 7 p.m.) and temperature (22–24 °C) conditions with free access to tap water and laboratory chow (BioBase Alimentação Animal, Santa Catarina, Brazil). All experiments were conducted between 7 and 12 a.m., and rats used in one experimental set were not reused in any other part of the study. The experimental protocols complied with the recommendations of the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the local Institution’s Animal Ethics Committee (CEUA-ICS-UFBA #026/2012). Experimental procedures were performed by trained personal, all efforts were employed to minimize animal suffering, and their health was monitored daily after surgical procedures.

Sodium depletion was achieved by subcutaneous injection of furosemide, a loop diuretic, 24 h prior to the experimental sessions. The animals had access to distilled water, 1.5% saline solution and standard rat chow from the guide cannula implant surgery until the time of furosemide administration. Access to 1.5% saline ceased immediately after the furosemide injection, and from this moment on, the animals continued to have free access to distilled water, while normal rat chow was replaced by a low sodium diet (0.001% Na+ and 0.33% K+), as previously published (Nascimento et al., 2014). Sodium depleted rats received i.c.v. injections of carvacrol or vehicle. Graded bottles containing 1.5% saline solution and distilled water were reintroduced into the cages immediately after central injections. Cumulative fluid intake was measured at 5, 10, 15, 20, 25, 30, 45 and 60 min after reintroduction of the bottles into the cages. To test the involvement of adrenergic pathways, rats received i.c.v. injections of prazosin (80, 160 and 320 μg) 30 min before carvacrol (300 μg) injections. In this experimental set there were, therefore, three control groups: 1) saline + vehicle; 2) prazosin (320 μg) + vehicle and 3) saline + carvacrol. In a different experimental set, the influence of brain serotonergic depletion in carvacrol effects was accessed by i.c.v. injections of 200 μg of pCPA 72 h before the experiment.

2.2. Surgical procedures

2.5. Open field test

The animals were anesthetized (ketamine/xylazine; 80/7 mg/kg i.p.) and placed in a stereotaxic apparatus with the skull leveled between the bregma and lambda. A guide cannula (22-gauge) was implanted into the left lateral ventricle (anteroposterior = −0.9 mm; lateral = +1.2 mm; vertical = −3.6 mm; relative to Bregma). These coordinates were based on The Rat Brain Atlas (Paxinos and Watson, 2009). The guide cannula was fixed to the skull with stainless steel screws and dental cement. An obturator was provided to avoid obstruction of the guide canula. After surgery, the animals were treated with an antibiotic combination of penicillin and streptomycin (Pentabiótico, Fort Dodge Ltda., Brazil; 0.2 ml/rat i.m.) and with the analgesic/anti-inflammatory agent, flunixin meglumine (2.5 mg/kg i.m.). The animals were then individually housed during 5 days for surgery recovery, with free access to laboratory chow, distilled water and 1.5% saline solution. Animals were handled every day to minimize the stress of the experimental procedure.

Locomotor activity was accessed in an open field test consisting of an acrylic cylinder (60 cm in diameter and 60 cm high walls) with an open top. The floor of the cylinder was divided into eight areas of equal size within 2 concentric circles (42.43 cm2). Different groups of sodiumdepleted rats received i.c.v. injections of carvacrol (150 and 300 μg) or 5 μL of corn oil 30 min before the open field test. Animals were carefully handled and placed in the center of the open area, a camera positioned above the apparatus recorded the sessions (10 min/rat), and the number of areas crossed (measured as the number of floor units entered with all four paws) was scored by a blinded experimenter to the procedures. The behavioral experiments took place in a sound-attenuated and temperature-controlled (22–24 °C) room. A white-noise generator provided constant background noise and the apparatus was cleaned with 70% ethanol and dried before each session to minimize olfactory cues. Animals were acclimatized into the experimental room 24 h prior to the test.

2.3. Drugs and microinjections

2.6. Immunohistochemistry

Central injections were given using a Hamilton microsyringe (10 μl) connected to an injectior through a polyethylene tubing. The total volume of 5 μl was slowly injected (120 s), with the injector remaining in the guide cannula for an additional 60 s. The drugs used were: carvacrol [5-Isopropyl-2-methylphenol; (CH3)2CHC6H3(CH3)OH], pCPA (4-ChloroDL-phenylalanine; tryptophan hydroxylase inhibitor) and prazosin [1-(4Amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furanylcarbonyl)piperazine hydrochloride; α-adrenoceptor antagonist], all of them purchased from Sigma-Aldrich (São Paulo, Brazil). Carvacrol was dissolved in corn oil (vehicle; VEH) and administered in the following quantities: 25, 50, 75, 150 and 300 μg/rat, while the control groups received 5 μL of vehicle only. Prazosin was used in the concentration of 80, 160 and 320 μg/rat, selected based on previous works (Colombari et al., 1990). The concentration of pCPA used was 200 μg/rat, based on previous studies (Hritcu et al., 2007). Rats received the same dose for all i.c.v. injected drugs, since brain size does not vary within the weight range of rats used.

Two independent rat cohorts were anesthetized (thionembutal, 40 mg/kg, i.p.; Abbott Laboratories; Illinois, USA) 30 min after i.c.v. injections of carvacrol (300 μg) or vehicle (corn oil), and transcardially perfused with 400 ml of phosphate buffered saline (PBS) 0.1 M (pH 7.4) followed by 4% paraformaldehyde (pH 7.4). Brains were carefully removed and stored overnight in the same fixative at 4 °C and then submerged in 30% sucrose solution for at least three days. The prosencephalic regions of the brain were serially sectioned at 40 μm in a cryostat. The free-floating sections of the brains were washed three times for 5 min in 0.01 M PBS, incubated in 1% hydrogen peroxide for 15 min to block endogenous peroxidase activity, washed a further three times in PBS and incubated in 5% normal goat serum for 1 h. Next, this solution was replaced by PBS with 0.3% Triton X-100 (Sigma Co., St. Louis, MO) and primary Fos antiserum diluted 1:4000 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at room temperature. The sections were rinsed with PBS and incubated with a biotinylated goat

2. Materials and methods 2.1. Animals

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anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA), diluted 1:400 in PBS for 1 h at room temperature, washed with PBS and finally incubated at room temperature for 60 min with avidin-biotin horseradish peroxidase complex (Avidin-Biotin Complex-Kit, Vector Laboratories, CA; 1:200). After the final three-wash steps, sections were incubated in a PBS solution containing 0.02% 3,3’-diaminobenzidinetetrahydrochloride (DAB; Sigma Co., St. Louis, MO), 0.08% nickel sulfate and 0.0002% hydrogen peroxide for 10 min. Sections were rinsed with PBS 5 min after DAB onset to stop the enzymatic peroxidase reaction. Immunohistochemistry was carried out simultaneously on the brains of experimental and control animals. Following these procedures, the brain sections were mounted on gelatin-coated glass slides and allowed to dry overnight, soaked for 20 min in 100% xylene and cover-slipped with Entellan (Merk, Damstadt, Germany). The number of Fos-immunoreactive (Fos-IR) nuclei was counted under a light-microscope using a computerized image analysis system (Image-Pro Plus, Media Cybernetic, Inc., Silver Spring, MD). Fos-IR was counted in one section of each area of interest, in each case the section that appeared to have the greatest number of Fos + nuclei. For bilateral structures, Fospositive nuclei were counted only from one side. An observer blinded to the treatment-groups quantified Fos + cells. Cells containing a nuclear brown-black reaction product were considered positive for Fos immunoreactivity. Neuroanatomical sites were identified with the help of Paxinos and Watson’s Atlas (Paxinos and Watson, 2009).

more effective in reducing saline consumption relative to the control group (60 min: VEH: 12.5 ± 0.8; CVC150: 3.0 ± 1.2; CVC300: 2.2 ± 0.9 ml; Two-way ANOVA, F(5,50) = 8.06, P < 0.0001; Fig. 1A). At the lower concentration of carvacrol employed (25 μg), salt intake was similar to the control group, and no statistical difference was found among them (60 min: CVC25: 9.9 ± 1.1 ml). The sodium intake in rats receiving 50 μg of carvacrol was significantly lower than the control group in the period of 20–30 min and at 60 min following microinjections, while at 75, 150 and 300 μM concentrations, the inhibitory effect was observed from 20 min after microinjections and lasted until the end of the experiments (Table 1). The cumulative water intake in sodium-depleted animals was very low and the i.c.v. carvacrol injections, in all doses used, did not significantly change water intake (Fig. 1B). Table 1 summarizes the data collected from all groups and time points accessed, and Table 2 summarizes the statistical results applied to this series of experiments. 3.2. Prior alpha-adrenergic blockade dose-dependently reduces the antinatriorexigenic effect of carvacrol Since the antinatriorexigenic effect of carvacrol was maximum at the highest concentration used, we injected 300 μg of carvacrol in the subsequent experiments. Similar to what was observed in the previous experiment, carvacrol injections at the concentration of 300 μg (n = 9) in animals pretreated with saline significantly inhibited salt appetite. The pretreatment with prazosin at the dose of 160 (n = 10) and 320 μg (n = 8) increased salt intake in carvacrol treated rats, reducing the inhibitory effect of carvacrol (60 min: PRA160μg + CVC300μg: 7.3 ± 1.4; PRA320μg + CVC300μg: 10.3 ± 1.2; SAL + CVC300μg: 1.7 ± 0.9 ml; Two-way ANOVA, F(5,45) = 12.03, P < 0.0001; Fig. 2A). Prazosin at the dose of 40 nM (n = 7) was unable to modify the inhibitory action of carvacrol on salt intake (Table 1). Carvacrol i.c.v. injections of 160 nM of prazosin plus vehicle did not change the salt intake induced by sodium depletion when compared with the control group treated with saline plus vehicle (60 min: PRA320μg + VEH: 10.5 ± 1.4; SAL + VEH: 12.9 ± 0.7 ml). The cumulative water intake was not altered by any of the treatments and remained low during all experiments (Fig. 2B). The salt solution intake for all time points are summarized in Table 1, and the statistical analysis summary can be seen in Table 2.

2.7. Histological procedures At the end of the experiments, animals were anesthetized with ketamine/xylazine (80/7 mg/kg i.p.) and given i.c.v. injections of 2% Evans blue dye at a volume of 2 μl. Five min later, they were submitted to transcardial perfusion with isotonic saline solution followed by 10% formalin. The brains were then removed and post-fixed in 10% formalin for 24 h at 4 °C. Brains were transferred to a 30% sucrose solution and maintained at 4 °C for at least 72 h for cryoprotection, before being sliced into 40 μm cryostat sections. To confirm the injection sites, the slices were stained with cresyl violet and analyzed at light microscopy. The surgery success in cannula implant within the lateral ventricle was above 90% (not shown), and data collected from animals in which the cannula missed the target were excluded from statistical analysis. 2.8. Statistical analysis

3.3. The antinatriorexigenic effect of carvacrol is suppressed in serotonergicdepleted rats

The data are presented as means ± standard error of the mean (S.E.M.). Statistical analyses were performed using the GraphPad Prism software (GPAD, version 6.03, San Diego, USA). The amount of water and sodium chloride solution consumed was analyzed separately using a mixed-model of two-way analysis of variance (ANOVA) with time as the within-subjects repeated measure (eight levels of time) and drug treatment as the between-subjects variable (not repeated). The Bonferroni post-hoc test was used to compare the effects of the drugs during each measurement period. Differences between the groups were considered statistically significant when p < 0.05. The data from the open field test were analyzed using one-way ANOVA followed by Newman-Keuls multiple comparisons test. For statistical analysis of the FOS expression data in brain areas, the non-parametric Mann-Whitney (MW) test was used. Differences between groups were considered statistically significant when p < 0.05.

Since carvacrol is also known to have inhibitory effects on serotonin reuptake (Mechan et al., 2011), we next decided to investigate if the antinatriorexigenic effect of carvacrol would persist in serotonin-depleted rats. Serotonin-depleted rats did not change sodium intake when compared to the control group receiving saline until the end of our experiments (pCPA + VEH: 12.9 ± 0.9; SAL + VEH: 12.6 ± 0.8 ml). As expected, the group of animals pretreated with saline which received carvacrol showed significant inhibition of salt intake. However, the inhibitory effect of carvacrol on salt appetite was impaired by the pretreatment with pCPA 60 min following carvacrol administration (pCPA + VEH: 12.9 ± 0.9; SAL + CVC300μg: 3.1 ± 1.2 ml). The cumulative water intake was similar in all groups (Fig. 3B). These results are summarized in Tables 1 and 2

3. Results

3.4. Carvacrol has no effects on locomotor activity

3.1. Central administration of carvacrol inhibits salt appetite in Na+depleted rats

Since the inhibition of sodium intake induced by carvacrol could be biased by changes in locomotor activity, we tested if central administration of carvacrol alters locomotion. Sodium-depleted rats receiving i.c.v. injections of carvacrol at the two highest concentrations in this study presented similar locomotor activity when compared to the group receiving vehicle, and no significant difference was noted (VEH:

Sodium-depleted rats presented a high sodium intake, and i.c.v. injections of carvacrol promoted a dose-dependent inhibition of salt appetite (Fig. 1A). Carvacrol at 150 and 300 μg concentrations was 121

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Fig. 1. Cumulative salt intake (A) and water intake (B) in sodium-depleted rats following carvacrol injections into the LV. Carvacrol dosedependently inhibited salt appetite in sodium depleted rats. *P < 0.05 relative to vehicle group; #P < 0.05 relative to the group receiving 33 μM of carvacrol. +P < 0.05 relative to the group receiving 67 μM of carvacrol. Data are presented as means ± S.E.M.

Table 1 Summary data of hypertonic saline (1.5%) intake in vehicle or CVC treated group (top), pretreated with prazosin (middle) or pretreated with pCPA (bottom). Statistical differences noted are relative to the VEH group. Data are presented as means ± S.E.M. a = P < 0.05; b = P < 0.01; c = P < 0.001. Experiment

Time (min) 5

CVC effect: VEH (n = 8) CVC25μg (n = 9) CVC50μg (n = 9) CVC75μg (n = 10) CVC150μg (n = 10) CVC300μg (n = 10) Prazosin pretreatment: VEH (n = 7) PRA320μg + VEH (n = 10) SAL + CVC300μg (n = 9) PRA80μg + CVC300μg (n = 7) PRA160μg + CVC300μg (n = 10) PRA320μg + CVC300μg (n = 8) pCPA pretreatment: SAL + VEH (n = 11) pCPA + VEH (n = 11) SAL + CVC300μg (n = 11) pCPA + CVC300μg (n = 8)

10

15

20

25

30 1.1 1.3 1.2a 1.1c 1.2c 0.8c

60

10.9 ± 1.0 9.4 ± 1.2 6.9 ± 1.6a 3.8 ± 1.3c 2.2 ± 1.2c 2.2 ± 0.9c

12.5 ± 0.8 9.9 ± 1.1 7.2 ± 1.6b 3.8 ± 1.3c 3.0 ± 1.2c 2.2 ± 0.9c

1.4 0.9 0.4 0.6 0.0 0.4

± ± ± ± ± ±

0.5 0.3 0.2 0.3 0.0 0.4

3.7 2.4 1.3 1.3 0.5 1.3

± ± ± ± ± ±

0.8 0.7 0.5 0.7 0.3 0.7

5.4 3.6 2.5 1.8 0.8 1.6

± ± ± ± ± ±

1.3 1.1 0.9 0.8 0.4a 0.8

7.2 5.3 3.1 2.1 1.2 1.6

± ± ± ± ± ±

1.3 1.3 1.0 1.0b 0.7c 0.8b

9.0 6.4 4.1 2.5 1.5 1.7

± ± ± ± ± ±

1.2 1.3 1.2a 1.0c 0.9c 0.8c

9.9 7.5 4.9 3.0 2.0 1.8

1.6 1.5 0.2 0.5 0.4 1.4

± ± ± ± ± ±

0.5 0.5 0.2 0.2 0.2 0.5

4.2 3.3 0.7 1.3 0.6 2.0

± ± ± ± ± ±

0.7b 1.0 0.4 0.6 0.2 0.7

6.1 4.8 1.0 1.6 1.3 3.9

± ± ± ± ± ±

1.1c 1.1 0.6 0.8 0.3 0.9

8.1 7.0 1.1 1.7 2.1 5.6

± ± ± ± ± ±

1.1c 1.4c 0.6 0.8 0.6 0.7a

9.9 8.6 1.2 2.0 3.9 6.9

± ± ± ± ± ±

1.0c 1.4c 0.7 1.0 1.1 0.7b

10.6 ± 1.0c 9.3 ± 1.4c 1.2 ± 0.7 2.4 ± 0.9 5.3 ± 1.4a 8.2 ± 0.9c

11.6 ± 0.8c 10.3 ± 1.4c 1.7 ± 0.8 3.3 ± 1.3 6.1 ± 1.4a 9.7 ± 1.1c

12.9 ± 0.8c 10.5 ± 1.4c 1.7 ± 0.9 3.3 ± 1.3 7.3 ± 1.4c 10.3 ± 1.2c

2.2 2.4 0.4 2.8

± ± ± ±

0.2 0.4 0.4 0.5

4.1 5.5 1.3 4.7

± ± ± ±

0.4 0.6c 0.7 0.7a

6.2 7.0 1.6 7.3

± ± ± ±

0.5c 0.8c 0.8 1.0c

7.6 8.8 1.6 9.4

± ± ± ±

0.7c 0.9c 0.8 1.0c

9.1 ± 0.8c 9.9 ± 0.8c 1.7 ± 0.8 10.8 ± 1.0c

10.5 ± 0.9c 10.6 ± 0.7c 1.8 ± 0.8 12.1 ± 0.9c

11.5 ± 0.7c 12.0 ± 0.8c 2.2 ± 0.9 13.0 ± 1.0c

12.6 ± 0.7c 12.9 ± 0.9c 3.1 ± 1.3 13.5 ± 0.8c

39.6 ± 6.3; CVC150μg: 37.8 ± 9.0; CVC300μg: 39.8 ± 4.6; ANOVA, F(2,11) = 0.03, P = 0.98; Fig. 4).

± ± ± ± ± ±

45

4. Discussion In the present study we found that i.c.v. injections of carvacrol significantly inhibited salt appetite in sodium-depleted rats. It was also observed that the antinatriorexigenic effect of carvacrol was abolished by brain serotonergic depletion and was reduced by alpha-adrenergic blockade. Since the locomotor activity of the animals treated with carvacrol was similar to the control group in the open field test, it is valid to conclude that the antinatriorexigenic effect of carvacrol is not due to any locomotor impairment induced by carvacrol, but rather, expressed as a consequence of changes in monoamine signaling. Importantly, previous studies in our group found that i.c.v. administration of carvacrol in the two highest concentrations employed in the present investigation was associated with a reduced blood pressure, without altering heart rate (Batista, 2017). Since a reduction in blood pressure is followed by increases in sodium intake (Turnhorst and Johnson, 1994), the effects of carvacrol in sodium appetite and hemodynamics are putatively independent from one another. Furthermore, carvacrol significantly increased the neuronal activation in brain areas involved in the control of salt appetite, such as the MnPO, OVLT, PVN, SON, CeA and MeA.

3.5. Carvacrol induces neuronal activation in prosencephalic areas involved in the control of salt intake To further elucidate how carvacrol could modulate sodium appetite behavior, we performed a cFos staining to access neuronal activation in areas involved in fluid homeostasis. Carvacrol induced a significant increase in the number of Fos + neurons in the organ vasculosum of the lamina terminalis (VEH: 40.8 ± 4.2; CVC300: 117.3 ± 13.95; MWtest, P = 0.029; Fig. 5), median preoptic nucleus (VEH: 90.0 ± 15.6; CVC300: 160.8 ± 6.6; MW-test, P = 0.016; Fig. 5), supraoptic nucleus (VEH: 52.7 ± 5.8; CVC300: 79.8 ± 8.3; MW-test, P = 0.032; Fig. 6), paraventricular nucleus (VEH: 60.8 ± 6.8; CVC300: 215.6 ± 19.3; MW-test, P = 0.016; Fig. 6), medial amygdala (VEH: 39.5 ± 4.0; CVC300: 107.6 ± 7.7; MW-test, P = 0.016; Fig. 7) and central amygdala (VEH: 26.3 ± 4.7; CVC300: 127.2 ± 6.3; MW-test, P < 0.016; Fig. 7) 30 min after i.c.v. injection at the highest concentration used in this study (300 μM; n = 5) when compared to vehicle. 122

European Journal of Pharmacology 854 (2019) 119–127

F(35,350) = 6.08 F(35,350) = 0.94 F(35,315) = 5.67 F(35,315) = 1.01 F(21,252) = 12.59 F(35,350) = 0.65

< 0.0001 = 0.5757 < 0.0001 = 0.4558 < 0.0001 = 0.8741

F(7,350) = 82.30 F(7,350) = 1.64 F(7,315) = 94.43 F(7,315) = 3.40 F(7,252) = 158.8 F(7,266) = 4.08

< 0.0001 = 0.1220 < 0.0001 = 0.0016 < 0.0001 = 0.0003

F(5,50) = 8.06 F(5,50) = 0.59 F(5,45) = 12.03 F(5,45) = 1.35 F(3,36) = 28.16 F(3,38) = 0.06

< 0.0001 = 0.7071 < 0.0001 = 0.2597 < 0.0001 = 0.9781

The control of sodium appetite is important to maintain the body homeostasis, and distinct brain areas and neurotransmitters are involved in the control of this behavior. Several investigations have suggested that serotonin possesses a major role in hidrosaline homeostasis, and that sodium appetite is increased in serotonin-depleted rodents (Walters, 1977; Lima et al., 2004; for review see Reis et al., 1994). In addition, studies have shown that serotonergic transmission plays an inhibitory role in sodium appetite, since body sodium overload increases serotonin availability within the hypothalamus (Munaro and Chiaraviglio, 1981). Peripheral administration of 5-HT1A and 5-HT2C receptors agonists inhibit salt intake in sodium-depleted rats (BadauêPassos et al., 2003; Cooper et al., 1988; Neill and Cooper, 1989). Furthermore, intracerebroventricular injections of 5-HT2C and 5-HT3 agonists in rats promote similar results (Castro et al., 2003). On the other hand, serotonergic depletion induced by peripheral administration of pCPA increases sodium intake in rats (Lima et al., 2004; Walters, 1977). In a screening of 200 pungent substances, carvacrol was previously identified as an agonist and potentiator of 5HT3A receptors (Ziemba et al., 2015). It has been shown that intraperitoneal administration of oregano´s extract inhibits monoamine oxidase (MAO) and inhibits the reuptake and degradation of serotonin in the hippocampus of rodents (Mechan et al., 2011). Thus, we presumed that the inhibition of sodium appetite induced by carvacrol may be, in some extent, mediated by an increased availability of serotonin in specific brain areas that lead to the inhibition of this behavior. This is supported by our data showing that the antinatriorexigenic effect of carvacrol was abolished in this particular group of animals. Besides changing brain serotonin, carvacrol may also influence other monoamine signaling. One study has shown that the administration of carvacrol over 7 days increased the dopamine and serotonin levels into the prefrontal cortex and the hippocampus (Zotti et al., 2013). In vitro studies have shown that carvacrol inhibits human noradrenaline transporters (Mechan et al., 2011), whereas increases the extracellular levels of serotonin and noradrenaline in vivo, which was associated with antidepressant and anxiolytic effects (Mechan et al., 2011). It has been previously shown that i.c.v. injections of NA and the alpha-adrenoreceptor agonist clonidine reduces sodium intake by 50–70% (De Luca et al., 2002). Thus, noradrenaline is likewise implicated in the regulation of the sodium appetite. In fact, intracerebroventricular administration of noradrenaline or alpha-adrenergic agonists inhibits salt intake induced by sodium depletion, water deprivation and angiotensin II administration (De-Luca and Menani, 1997; De Paula et al., 1996; Sato et al., 1996; Sugawara et al., 1999; Yada et al., 1997). We reasoned that carvacrol could also inhibit sodium intake in sodium-depleted animals by means of alpha-adrenergic receptor activation. In order to test this hypothesis, carvacrol was administered in animals pretreated with prazosin, a selective alpha1adrenergic receptor antagonist. It was observed that prazosin reduced the antinatriorexigenic effect of carvacrol, and the inhibitory effect of prazosin was exerted in a concentration-dependent manner. Therefore, these data suggest that the inhibitory effect of carvacrol on sodium appetite may also depend on alpha-adrenergic receptors. The antinatriorexigenic effect of carvacrol observed here does not seems to be due to locomotor deficit, since in the open field test, animals receiving the highest dose of carvacrol (400 μM) presented similar locomotor activity to the control group. Our findings are in accordance with literature data, since it has been previously reported that mice receiving oral administration of carvacrol does not display any changes on locomotor activity in the open field test (Melo et al., 2010). Different brain areas participate in the control of sodium appetite. The circumventricular organs, such as OVLT and SFO, present an arrangement of the blood-brain barrier that allows an easy exchange of substances between peripheral and brain substances (Duvernoy and Risold, 2007; Mckinley et al., 1990). Furthermore, ependimary cells and astrocytes within the OVLT, SFO and MnPO present angiotensin II receptors and sodium channel receptors (Na+ channels) that make

CVC effect Salt intake Water intake Prazosin pretreatment Salt intake Water intake pCPA pretreatment Salt intake Water intake

P F(DFn,DFd) F(DFn,DFd) P F(DFn,DFd)

P

Drug Time Interaction (Time x Drug) Experiments

Table 2 The amount of water and hypertonic saline solutions consumed was analyzed using a mixed-model analysis of variance (ANOVA), with one repeated measure factor (8 levels of time) and one group factor (drugs) not repeated. The post-hoc Bonferroni test was used to compare the treatment (drug) effect at each time point, as shown in Figs. 1–3.

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Fig. 2. Blockage of alpha1-adrenoreceptors inhibits the antinatriorexigenic effect of CVC in sodium-depleted rats. A) Pretreatment with prazosin inhibits, in a dose-dependent manner, the CVC effects on sodium appetite. B) No effect was seen in the water intake in all groups studied. *P < 0.05 relative to the SAL + VEH group. #P < 0.05 relative to the SAL + CVC 400 μM group. +P < 0.05 relative to the PRA160nM + VEH group. Data are presented as means ± S.E.M.

these areas important sites for peripheral information concerning sodium balance access to the brain (Hiyama et al., 2002; McKinley et al., 2003; Noda and Hiyama, 2015). These areas are interconnected among themselves and also project to the PVN and SON, which control vasopressin secretion and, consequently, regulate the water balance (Anderson et al., 2001; Lind et al., 1982; McKinley et al., 2015; Miselis et al., 1979). In our investigation, carvacrol was able to increase the neuronal activation in brain areas such as the MnPO, OVLT, PVN, SON. Interestingly, all these above mentioned activated brain areas by carvacrol in our study have been shown to increase FOS + neurons during satiety process of sodium appetite induced by peritoneal dialysis in rats, associating the activation of these brain areas with the inhibition of sodium appetite (Franchini and Vivas, 1999). This is further supported by later works from the same group showing that the SON and PVN cells are not activated by peritoneal dialysis per se (Franchini et al., 2002), suggesting that their activation participates, specifically, in the down-regulation of sodium intake. Other nuclei that showed Fos-IR by carvacrol administration were CeA and MeA. These structures are also part of the brain circuitry regulating sodium appetite. It was demonstrated that electrolytic lesion of CeA abolishes salt appetite induced by different models of salt intake (Galaverna et al., 1992; Zardetto-Smith et al., 1994). CeA and MeA are interconnected, and CeA is the main nucleus of the amygdaloid

complex that sends output to brain areas related to salt appetite control such as PVN, MnPO, as well as the bed nucleus of stria terminalis (Sah et al., 2003; Volz et al., 1990). It has been demonstrated that both serotonergic receptors and adrenergic receptors present in the amygdaloid complex nuclei participate in the control of salt appetite (Andrade-Franzé et al., 2010; Luz et al., 2006, 2007). Activation of 5HT3, but not 5-HT2C receptors within the CeA and MeA significantly reduced salt intake in sodium-depleted rats, an effect abolished by the pretreatment with the selective 5-HT3 receptor antagonist ondansetron (Luz et al., 2006, 2007). Carvacrol-induced activation of neurons within both sub-regions of the amygdala suggests a possible activation of 5HT3 receptors postsynaptically located as a secondary consequence from an increased availability of serotonin within these brain regions, a mechanism that could partially explain carvacrol natriorexigenic effect in the present study. In the present investigation, we found that carvacrol antinatriorexigenic effect was abolished under serotonin depletion. Arguably, following serotonin depletion, carvacrol activity in sodium intake would putatively depend on the final balance of carvacrol effects via noradrenergic signaling. Noradrenaline i.c.v. administration has been associated with both increase (Chiaraviglio and Taleisnik, 1969) and decrease (de Paula et al., 1996; Yada et al., 1997) in sodium intake, which reflects the wide range of noradrenaline effects through different Fig. 3. Serotonin depletion impairs the antinatriorexigenic effect of carvacrol in sodiumdepleted rats. A) Cumulative salt intake comparison shows that the pretreatment with pCPA inhibit the antinatriorexigenic effect of CVC administered in the highest concentration used in this study (400 μM). B) No changes in water intake were observed for all groups and time points studied. Data are presented as means ± S.E.M. *P < 0.05 relative to the SAL + CVC400μM group.

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agonist, prazosin also binds alpha2-receptors, which could occur at a higher extent when employed at elevated concentrations (Nahorsky et al., 1985). Serotonin can also exert opposing effects on sodium intake according to the brain region and specific receptor subtype activation. I.c.v. administration of ketanserin, a 5-HT2 antagonist, was associated with a reduced saline intake in sodium depleted rats (Gentili et al., 1991), but a similar effect was seen by mCPP (5-HT2 agonist) injection in the 3rd ventricle (Castro et al., 2003). In the LPBN, blockage of 5HT2 receptors was shown to increase sodium intake (Menani et al., 1998), whereas activation of 5-HT1A receptors within this nucleus increased sodium appetite (De Gobbi et al., 2005). Activation of 5-HT2 receptors within the LPBN inhibited sodium ingestion, while activation of 5-HT3 within this nucleus increased salt intake in sodium depleted rats (De Gobbi et al., 2007). Opposingly, 5-HT3 agonist injections in the central or medial amygdala were associated with antinatriorexigenic effect (Luz et al., 2006, 2007). In the PVN, serotonin or activation of 5HT1A receptors by 5-OH-DPAT inhibited sodium appetite (Villa et al., 2007). 5-HT1A receptors are mainly presynaptically located and exert negative feedback in the serotonin release. Thus, activation of these receptors in certain brain nuclei could decrease serotonin release from presynaptic terminals, suggesting that a reduced availability of serotonin in the LPBN increases sodium intake, whereas serotonin reduction is associated with a lower salt appetite in the PVN. Further, activation of 5-HT3 receptors in the LPBN and amygdala can increase or reduce, respectively, sodium intake. Therefore, our data suggest that carvacrol antinatriorexigenic activity reflects the final balance of the alterations of both serotonin and noradrenaline signaling, which is computed by different homeostasis-related brain nuclei. This behavioral effect promoted by carvacrol is altered either by central alphanoradrenergic blockage or serotonin depletion, suggesting that carvacrol activity on sodium intake depends on the integrity of both noradrenergic and serotonergic brain signaling. Taken together, the findings of the present study show that central administration of carvacrol exerts inhibitory actions on the control of salt appetite through the activation of both serotoninergic and alphaadrenergic pathways. Moreover, our work provides evidence of a cellular resolution of carvacrol activity in the brain. The antinatriorexigenic effect of carvacrol may depend on the activity of homeostasis-related brain areas, such as the MnPO, OVLT, PVN, SON, CeA and MeA, and future works should focus in elucidating the precise mechanisms by which monoamine signaling could be altered by carvacrol within these regions. Finally, our investigation contributes to the body of evidence placing carvacrol as a potential therapeutic compound, suggesting mechanisms and brain areas that could be involved in its protective properties.

Fig. 4. Number of areas crossed over a 10 min period in the open field test by sodium-depleted rats receiving i.c.v. injections of vehicle or the two-highest carvacrol concentrations used in this study. Data are expressed as means ± S.E.M. There was no statistically significant difference among the experimental groups.

receptors in homeostasis-related brain areas. These findings leaded researchers to develop a “dual-role hypothesis”, in which noradrenaline signaling participates in both increased and decreased sodium intake (Yada et al., 1997; Fitzsimons, 1998). This hypothesis is further supported by studies showing that clonidine, an alpha2-adrenergic agonist, reduced saline intake when injected into the medial septal area (Yada et al., 1997), but increased sodium appetite when injected into the hypothalamus (Almeida et al., 1999). Interestingly, prazosin injected into the hypothalamus also increased sodium intake, while yohimbine and propranolol, alpha2-and beta-adrenergic antagonists, respectively, exerted antinatriorexigenic effects (Almeida et al., 1999). In the PVN, prazosin decreases whereas yohimbine (alpha2-adrenergic antagonist) or noradrenaline increases sodium intake (Camargo and Saad, 2001, Camargo et al., 2003). Interestingly, sodium appetite in this nucleus can be reduced or enhanced by alpha1a- and alpha1b-adrenergic antagonists, respectively. Reduced and enhancement of sodium appetite was also seen after beta1-and beta2-adrenergic antagonists (Camargo et al., 2003). Finally, injections of noradrenaline within the lateral parabrachial nucleus (LPBN) also increases saline intake in sodium depleted rats (Gasparini et al., 2009). When taken together, these works support the hypothesis that upon serotonin depletion, carvacrol effects through noradrenergic pathways might not be enough to exert its antinatriorexigenic effects. In addition, carvacrol antinatriorexigenic effect was nearly eradicated under blockage of alpha-adrenergic receptors at a high concentration. Although mostly employed as an alpha1-adrenergic inverse

Fig. 5. Carvacrol i.c.v. injections increase FOS expression within the MnPO and OVLT nuclei. A) Representative micrographies (40×) of transversal slices containing the MnPO nucleus (top) and OVLT nucleus (bottom) for both groups, according to Paxino’s Atlas (right). B) Rats receiving CVC i.c.v. injections displayed statistically higher FOS-IR nuclei within the MnPO and OVLT when compared with the control group receiving vehicle. Data are presented as means ± S.E.M. *P < 0.05.

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Fig. 6. Carvacrol i.c.v. injections increase FOS expression within the SON and PVN nuclei. A) Representative micrographies (40×) of transversal slices containing the SON nucleus (top) and PVN nucleus (bottom) for both groups, according to Paxino’s Atlas (right). B) Rats receiving CVC i.c.v. injections displayed statistically higher FOS-IR nuclei within the SON and PVN when compared with the control group receiving vehicle. Data are presented as means ± S.E.M. *P < 0.05.

Fig. 7. Carvacrol i.c.v. injections increase FOS expression within the MeA and CeA nuclei. A) Representative micrographies (40×) of transversal slices containing the MeA nucleus (top) and CeA nucleus (bottom) for both groups, according to Paxino’s Atlas (right). B) Rats receiving CVC i.c.v. injections displayed statistically higher FOS-IR nuclei within the MeA and CeA when compared with the control group receiving vehicle. Data are presented as means ± S.E.M. *P < 0.05.

Funding and disclosures

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