Injections of the α-2 adrenoceptor agonist clonidine into the dorsal raphe nucleus increases food intake in satiated rats

Journal Pre-proof Injections of the α-2 adrenoceptor agonist clonidine into the dorsal raphe nucleus increases food intake in satiated rats Rafael Appel Flores, Renata Steinbach, João A.B. Pedroso, Martin Metzger, José Donato, Jr., Marta Aparecida Paschoalini PII:

S0028-3908(20)30465-2

DOI:

https://doi.org/10.1016/j.neuropharm.2020.108397

Reference:

NP 108397

To appear in:

Neuropharmacology

Received Date: 6 June 2020 Revised Date:

4 November 2020

Accepted Date: 7 November 2020

Please cite this article as: Flores, R.A., Steinbach, R., Pedroso, Joã.A.B., Metzger, M., Donato Jr., , José., Paschoalini, M.A., Injections of the α-2 adrenoceptor agonist clonidine into the dorsal raphe nucleus increases food intake in satiated rats, Neuropharmacology (2020), doi: https://doi.org/10.1016/ j.neuropharm.2020.108397. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author contributions RF and MP conceived the study and designed experiments. RF performed all the experiments with the participation of RS in the behavioral tests. RF, JP and JD performed immunohistochemical and immunofluorescence analyzes, and JD supplied reagents and materials. MM contributed to the image collection and data analysis. RF wrote the manuscript with input from all other authors and revision from MP, MM, and

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Injections of the α-2 adrenoceptor agonist clonidine into the dorsal raphe nucleus increases food intake in satiated rats

Rafael Appel Flores *1, Renata Steinbach1, João A.B. Pedroso2, Martin Metzger 2, José Donato Jr.2, Marta Aparecida Paschoalini1. 1

Department of Physiological Sciences, Center of Biological Sciences - CCB, Federal

University of Santa Catarina (UFSC), 88040-970 Florianópolis, SC, Brazil.

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of São Paulo, 05508-900 São Paulo, SP, Brazil.

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Number of manuscript figures: 3 Number of supplementary figures: 2

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Number of manuscript tables: 1

Number of supplementary tables: 1

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Number of pages: 24

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Department of Physiology & Biophysics, Institute of Biomedical Sciences, University

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* Corresponding author: Rafael Appel Flores

E-mail address: [email protected] Telephone: +55 48 99626-0817 Fax number: +55 48 3721-9672

Authors: Renata Steinbach: [email protected] João A.B. Pedroso: [email protected] Martin Metzger: [email protected] Jose Donato Jr.: [email protected] Marta Aparecida Paschoalini: [email protected]

Abstract The present study aimed to evaluate the effects of pharmacological manipulation of αadrenergic agonists in the dorsal raphe nucleus (DR) on food intake in satiated rats. Adult male Wistar rats with chronically implanted cannula in the DR were injected with adrenaline (AD) or noradrenaline (NA) (both at doses of 6, 20 and 60 nmol), or α-1 adrenergic agonist phenylephrine (PHE) or α-2 adrenergic agonist clonidine (CLO) (both at doses of 6 and 20 nmol). The injections were followed by the evaluation of ingestive behaviors. Food and water intake were evaluated for 60 minutes. Administration of AD and NA at 60 nmol and CLO at 20 nmol increased food intake

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and decreased latency to start consumption in satiated rats. The ingestive behavior was

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not significantly affected by PHE treatment in the DR. CLO treatment increased Fos

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expression in the arcuate nucleus (ARC) and paraventricular nucleus of the hypothalamus (PVN) in rats that were allowed to eat during the experimental recording

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(AF group). However, when food was not offered during the experiment (WAF group),

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PVN neurons were not activated, whereas, neuronal activity remained high in the ARC when compared to control group. Noteworthy, ARC POMC neurons expressed Fos in

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the AF group. However, double-labeled POMC/Fos cells were absent in the ARC of the WAF group, although an increase in Fos expression was observed in non-POMC cells

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after CLO injections in the WAF group. In conclusion, the data from the present study

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highlight that the pharmacological activation of DR α-adrenoceptors affects food intake in satiated rats. The feeding response evoked by CLO injections into DR was similar to that induced by NA or AD injections, suggesting that the hyperphagia after NA or AD treatment depends on α-2 adrenoceptors activation. Finally, we have demonstrated that CLO injections into DR impact neuronal activity in the ARC, possibly evoking a homeostatic response toward food intake. Keywords: Serotonin, POMC, Arcuate nucleus, noradrenaline, clonidine, Food intake.

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Introduction The dorsal raphe nucleus (DR) is a heterogeneous cluster of neurons located along

the midline of the brainstem and is the main source of serotonergic projections to the prosencephalon (Jacobs and Azmitia, 1992; Ren et al., 2019). This nucleus sends projections to many forebrain structures, including a dense innervation to the hypothalamus (Muzerelle et al., 2016; Vertes and Linley, 2008). The effect of serotonin (5-HT) on feeding behavior is already well established and has been mainly associated with the satiety process (Blundell, 1991; Blundell and Latham, 1979). There is manifold evidence that 5-HT action on food intake involves hypothalamic neurons of the arcuate nucleus (ARC), specially pro-

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opiomelanocortin (POMC) neurons and agouti-related peptide (AgRP) neurons (Heisler, et

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al., 2003; Heisler, et al., 2006; Garfield et al., 2015).

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DR neural activity is modulated by catecholamines, with DR receiving robust inputs from several noradrenergic cell groups, such as the caudoventrolateral medula (A1),

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commissural part of the nucleus of the solitary tract (A2), locus coeruleus (A6), and the A5

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noradrenergic cell group (Peyron et al., 1996). As demonstrated by in situ hybridization studies, DR also displays high levels of α-1 adrenoceptors mRNAs in 5-HT neurons (Day et

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al., 1997; Day et al., 2004; Pieribone et al, 1994). Immunohistochemical and autoradiography studies described a moderate presence of α-2a and α-2c adrenoceptor in the

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DR (Rosin et al., 1993; Talley et al., 1996; Unnerstall et al., 1984). However, in situ

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hybridization failed to reveal a significant density of the mRNAs encoding α-2 adrenoceptors in the DR, suggesting that the majority of these receptors are located presynaptically on noradrenergic terminals (McCune et al., 1993; Nicholas et al., 1993; Scheinin et al., 1994). Electrophysiological and microdyalisis experiments disclosed that endogenous noradrenaline (NA) exerts a stimulatory control on 5-HT release in the DR, through the activation of postsynaptic α-1 adrenoceptors and an inhibitory influence via activation of presynaptic α-2 autoreceptors (Baraban and Aghajanian, 1980; Bortolozzi and Artigas, 2003; Pudovkina, et al., 2003; VanderMaelen and Aghajanian, 1983). Previous pharmacological (Anderberg et al., 2017; Carlini et al., 2004), chemogenetic (Stachniak et al., 2014) and optogenetic studies (Nectow, et al., 2017) have documented an intricate influence of the DR in controlling food intake, suggesting that specific populations of DR neurons can either increase or suppress feeding (Nectow, et al., 2017). Thus, the optogenetic or chemogenetic activation of GABAergic DR neurons has been shown to increase food intake, while activation of glutamatergic DRN neurons

suppresses feeding (Nectow, et al., 2017). In line with this, injections of 5-HT or 8-OHDPAT, a 5-HT1A receptor agonist, into the DR induce feeding in satiated rats probably via activation of inhibitory DR 5-HT1A somatodendritic autoreceptors, that may regulate the release of 5-HT in its projections (Fletcher and Davies, 1990; Hutson et al., 1986). In a recent study, we demonstrated that DR is under the influence of postprandial signals generated during feeding (Flores et al., 2018). Moreover, comparisons between fasting animals and animals submitted to refeeding disclosed that the number of Fos positive neurons increased in DR after food intake (Wu et al., 2014). Based on this scenario, we hypothesized that the injection of α-adrenoceptor agonists into DR may affect ingestive

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responses in fed animals. Accordingly, this study aims to evaluate in free feeding rats, the effects of pharmacological manipulations of α-adrenergic agonists in the dorsal raphe

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nucleus (DR) on food intake, together with the impact of these manipulations on the

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neuronal activity of hypothalamic nuclei involved in the control of appetite and satiety.

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Materials and methods

2.1

Animals and surgical procedures Male Wistar rats were housed in groups of five in a temperature-controlled (21 ± 2

ºC) room. Standard rodent chow and water were provided ad libitum on a 12/12 h light/dark cycle (lights on at 7:00 am). All experimental procedures in this study were performed following the ethical principles of animal experimentation, postulated by the Brazilian College of Animal Experimentation, and the trial protocol approved by the Ethics Committee on the Use of Animals of the Federal University of Santa Catarina (CEUA protocol: PP0075). All efforts were made to minimize the number of animals used and their

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pain and discomfort.

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After anesthesia with a mixture of xylazine (13 mg kg-1) and ketamine (87 mg kg-1)

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injected intraperitoneally, animals weighing between 280 and 300g underwent stereotaxic surgery for implantation of guide cannula for subsequent drug microinjection into the DR.

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The stainless steel guide cannula (30 G) was implanted about 2 mm dorsolateral to DR in

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order to not injure the DR proper, according to the coordinates (anteroposterior to bregma: + 7.9 mm, lateral: + 2.2 mm and dorsoventral: - 4.8 mm) as described by Paxinos and Watson

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(2007). The cannula was anchored to the skull with jeweler’s screws and fixed with dental cement. To prevent rupture of the superior sagittal sinus and obstruction of the cerebral

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aqueduct during stereotaxic surgery, the stem was tilted 20º.

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After surgery, the rats were housed in groups of five with free access to food and water for one week for post-surgical recovery. Next, rats were kept in individual boxes and received food and water ad libitum until the day of the experiment.

2.2

Drugs and injections The adrenergic agonist, adrenaline (AD), and noradrenaline (NA) (Sigma Chemical

Co., USA) were injected at doses of 6, 20, and 60 nmol. The α-1 adrenergic agonist phenylephrine (PHE) and the α-2 adrenergic agonist clonidine (CLO) (Tocris, USA) were injected at doses of 6 and 20 nmol. A sterile solution of 0.9% NaCl was used as a vehicle for drug dilution or injected alone in the control groups (0.2 µL, VEH). The drug doses used were based on previous studies of our research group (Mansur et al., 2010, dos Santos et al., 2009). Drug or vehicle injections were performed using a 33 G needle 2 mm longer than the guide cannula connected to 1 µL SGE® syringe. The solutions were administered during 60

s, followed by a further 60 s with the needle still inside the guide cannula for better diffusion of the solution.

2.3

Experimental procedures After the post-surgical period, the rats were habituated to the recording chamber for

two consecutive days (60 min each day) before the experimental session. The experimental procedures were divided into two stages: Stage 1) Evaluation of ingestive behaviors after injection of AD, NA, PHE or CLO into the DR of satiated animals. At this stage, separate animals were used across doses, in which each animal received a single injection of drug or

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vehicle. Immediately after microinjections, the animals were placed in a recording chamber containing rodent pellet chow (Nuvilab CR-1, regular diet: 3.85 kcal/g, 10% kcal fat, 20%

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kcal protein, and 70% kcal carbohydrate; Nuvital, Brazil) in a feeder and water in a bottle

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placed outside the test chamber with a spout projected through the wall of the chamber. The

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digital recording of the session (60 min) was initiated with a webcam perpendicularly located 60 cm above the recording chamber floor, and the amount of food and water intake

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was recorded by the difference between food or water weight at the beginning and at the end of the recording period. At the end of the recording period, any food that occasionally

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spilled on the cage floor was recovered and weighed with the food that remained in the

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feeder. The recoding chamber has measures of length and width similar to those of the hosting cages (49 X 34 cm), however, having the largest height measure (40 cm) to prevent

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escapes. The recording chamber was coated with a black adhesive plastic paper to facilitate behavioral recording. A researcher blinded to the experimental groups was designated to perform the behavioral analysis using EthoLog 2.2.5 software to analyze the video-recorded data (Ottoni, 2000). The variables analyzed for food intake were the amount of chow consumed, the latency to start the behavior (in seconds), the frequency (number of episodes that the animal exhibited the feeding behavior), and the total duration of behavior (in seconds) during the 60 min of recording. For water intake, only the amount of water drunk was analyzed. Stage 2) The most effective PHE or CLO doses that affected food intake in stage 1 were injected into DR of new experimentally naive satiated rats with access to food during the experiment (access to food group – AF) or that have no access to food during the behavioral recording (without access to food group –WAF). This experiment aimed to study neuronal activity by Fos protein expression in hypothalamic nuclei that are critically involved in the regulation of food intake and energy balance. The WAF group was used to

exclude the influence of post-prandial signals induced by food intake on Fos expression. Importantly, these experimental procedures were always started one hour after the lights turned on, from 8:00 am to 10:00 am (light cycle). In spite of the fact that rats are mostly nocturnal feeders, this condition was chosen, according to one of the principal aims of the study to induce hunger even in a fed (satiated) animal. 2.4

Histological confirmation of drug injection site For the dose/response curve at the end of each experiment the animals were

anesthetized with a mixture of xylazine (13 mg kg-1) and ketamine (87 mg kg-1) injected

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intraperitoneally and then transcardially perfused with saline (0.9%) followed by formalin (10%). Brains were cut in 50 mm thick sections, in the coronal plane, using a cryostat. The

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sections were stained with cresyl violet and the position of the injection was assessed on a

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light microscope. The Paxinos and Watson rat atlas (2007) was used for evaluation of the

Peroxidase immunohistochemistry

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injection sites (DR).

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Ninety minutes following drug or vehicle injections, the animals were anesthetized with a mixture of xylazine (13 mg kg-1) and ketamine (87 mg kg-1) injected

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intraperitoneally and perfused transcardially with 0.9% saline solution followed by

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formaldehyde solution (prepared from 4% paraformaldehyde). Brains were collected and post fixed in the same fixative for 1–2 h and cryoprotected for two days at 4 ºC in 0.1 M PBS, containing 20% sucrose. Brains were cut at 40 µm in the coronal plane using a freezing microtome. Four series of tissue slices were collected in a cryo-protectant solution and stored at - 20 ºC. Brain sections were washed in a 0.02 М potassium PBS, pH 7.4 solution (KBPS) for 30 minutes for the complete removal of antifreeze solution. Sections were then incubated in a KPBS solution containing 0.3% Triton X-100 (Sigma Chemical, St. Louis, MO, USA; KBPS-T solution) and 0.3% hydrogen peroxide for 30 minutes. The sections were washed again in KBPS and incubated for 60 minutes in KPBS-T containing 3% normal donkey serum to block non-specific antibody binding. Subsequently, sections were incubated overnight with anti-Fos primary antibody (1:20,000; Calbiochem, EMD Chemicals Inc., San Diego, USA) made in rabbit containing KPBS-T with azide and 3% donkey normal serum at room temperature. Sections were then incubated for 60 minutes with a KPBS-T solution

containing a biotinylated anti-rabbit secondary antibody (1:1,000; Jackson ImmunoResearch Inc., West Grove, PA, USA). Then, the sections were incubated for 60 minutes in KPBS containing the avidin-biotin complex (ABC; 1: 5000; Vector Laboratories, Inc. Burlingame, CA, USA). The sections were then subjected to a reaction using 0.1 M acetate buffer, diaminobenzidine (DAB; Sigma Chemical, St. Louis, MO, USA), 0.5% nickel sulfate and 0.01% hydrogen peroxide. The sections were finally mounted onto gelatin-coated slides and covered using DPX (Sigma Chemical, St. Louis, MO, USA).

2.6

Double immunofluorescence staining

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All subsequent washes and incubation steps were performed under gentle agitation

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and protected from illumination. In order to identify whether Fos is expressed in neurons of

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the arcuate nucleus of the hypothalamus that express the bioactive protein α-melanocytestimulating hormone (α-MSH), double immunofluorescence staining was performed for α-

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MSH and Fos protein (α-MSH/Fos). The sections were washed in KPBS for 30 minutes,

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followed by incubation for 60 minutes in KPBS-T containing 3% normal donkey serum (Jackson Immunoresearch). Then, brain sections were incubated for 48 hours at 4°C in a

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cocktail of two primary antibodies: anti-Fos made in rabbit (1:10,000; Calbiochem) and antiα-MSH made in sheep (1:2000; Chemicon, Cat. AB5087). Next, the sections were incubated

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for 60 minutes with a cocktail of fluorescent secondary antibodies (all from Jackson

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Immunoresearch) conjugated with Alexa Fluor 488 (1:500; anti-rabbit) and Alexa Fluor 594 (1:500; anti-sheep). After incubation, the sections were washed in KPBS and mounted on gelatinized slides. The slides were covered with Fluomount G mounting medium (E.M.S.). 2.7

Image collection and data analysis Immunoperoxidase and immunofluorescence reactions were analyzed on an

Axioimager A1 microscope (Zeiss, Muenchen, Germany) for the identification of Fos+ cells after peroxidase reaction or α-MSH-only and α-MSH+/Fos+ cells after double immunofluorescence reaction. Digital photomicrographs were taken from representative sections using a Zeiss Axiocam HRc (Zeiss) camera. Images of double immunofluorescent labels were acquired with the aid of Axiovision software (Zeiss), which allows the acquisition of images from different fluorescent channels, as well as the overlap of images. Three representative sections of the analyzed nucleus of each animal were selected. For the counts, the Image J 1.46r software was used, considering the whole area of the nucleus in

each section. This analysis was performed by a researcher with experience in counting Fos immunoreactivity which was blinded to the experimental groups. The number of Fos+ cells were quantified in the following hypothalamic nuclei: Paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), arcuate nucleus (ARC) and lateral hypothalamic area (LHA), as well as in the basolateral amygdala (BLA). The number of α-MSH+/ Fos+ cells was quantified in the ARC. Quantified nuclei were identified, named, and had their limits established according to the rat brain atlas of Paxinos and Watson (2007). Specifically in the ARC, an additional analysis was carried out considering the different subregions present in this nucleus. Fos+ cells were quantified in the ventromedial (VM), dorsomedial (DM), and

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ventrolateral (VL) divisions of the ARC, as previously described by Kirk et al. (2017). Image processing was performed using Adobe Photoshop software (Adobe Systems,

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Mountain View, CA, version 2015) by adjusting only the color balance, contrast, and

Statistical analysis

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brightness of the images.

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The dose/response curves of ingestive behaviors were analyzed by analysis of variance (ANOVA) followed by Tukey post hoc analysis. For the immunohistochemistry

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groups, the number of labeled cells per nuclei was analyzed separately, first by the

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Kolmogorov-Smirnov test to assess normality, then by the unpaired Student’s t-test or ANOVA test followed by Tukey post hoc analysis. In all statistical analyses, only p values <

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0.05 were accepted as statistically significant. The statistical analysis was performed with the Statistica 8.0 software (STATSOFT, Tulsa, OK, USA). 3

Results All animals included for statistical analysis (n = 112 rats) had the accuracy of the

injection site in the DR confirmed after histological analysis (Figure 1A-B).

3.1

Changes in feeding behavior after injection of adrenaline (AD) into the DR of

satiated rats. The AD injection of 60 nmol dose into the DR increased food intake in satiated rats [F (3, 24) = 18.73 p < 0.0001] (Figure 1C), as well as feeding frequency [F (3, 24) = 21.12 p < 0.0001] and duration [F (3, 24) = 21.27 p < 0.0001] when compared with the control group (vehicle injection; Table 1). The latency to start feeding [F (3, 24) = 12.94 p < 0.0001] decreased after the administration of AD 60 nmol (Table 1). Lower doses of AD did not

affect ingestive behaviors. Animals injected with AD, NA, PHE, CLO or vehicle did not drink water during the evaluated period (data not shown).

Feeding Duration

Feeding Frequency

Feeding Latency

VEH 6 nmol 20 nmol 60 nmol

0 38 ± 30 59 ± 39 279 ± 17 *

0 0.4 ± 0.3 0.4 ± 0.3 3.3 ± 0.5 *

3600 ± 0 2813 ± 502 2924 ± 443 823 ± 43 *

NA

VEH 6 nmol 20 nmol 60 nmol

0 0 25 ± 17 637 ± 66 *

0 0 0.2 ± 0.2 4 ± 0.6 *

3600 ± 0 3600 ± 0 2900 ± 494 861 ± 102 *

PHE

VEH 6 nmol 20 nmol

15 ± 15 62 ± 43 21 ± 21

0 0.5 ± 0.3 0.1 ± 0.1

3417 ± 183 3226 ± 267 3536 ± 64

CLO

VEH 6 nmol 20 nmol

23 ± 23 54 ± 41 640 ± 28 *

0.1 ± 0.1 0.3 ± 0.2 6 ± 0.5 *

3394 ± 206 3112 ± 330 609 ± 65 *

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AD

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Dose

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Drug

Changes in feeding behavior after injection of noradrenaline (NA) into the DR of

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Table 1: Feeding Duration, Feeding Frequency and Feeding Latency during 60 minutes of experimental recording after the injection of adrenaline (AD), noradrenaline (NA), phenylephrine (PHE), clonidine (CLO) or vehicle (VEH) into DR of satiated rats. In all set of experiments, separate animals were used across doses, in which each animal received a single injection of drug or vehicle. Data represent the mean ± SEM * p < 0.05 vs vehicle group. Non-paired one-way ANOVA followed by Tukey's post hoc test (n = 6 8 per group).

satiated rats

In satiated rats, NA injection into the DR in the 60 nmol dose increased food intake [F (3, 23) = 27.04 p < 0.0001] (Figure 1D), feeding frequency [F (3, 23) = 51.29 p < 0.0001] and feeding duration [F (3, 23) = 102.0 p < 0.0001] (Table 1). The latency to start feeding [F (3, 23) = 38.70 p < 0.0001] decreased after administration of AD 60 nmol (Table 1). All other doses of NA did not affect ingestive behaviors. 3.3

Changes in feeding behavior after injection of phenylephrine (PHE) into the DR of

satiated rats The ingestive behaviors were not significantly affected by PHE injection into the DR at both 6 and 20 nmol doses (Figure 1E and Table 1).

3.3 Changes in feeding behavior after injection of clonidine (CLO) into the DR of satiated rats The injection of CLO 20 nmol increased food intake [F (2, 18) = 21.36 p < 0.0001] (Figure 1F), feeding frequency [F (2, 18) = 104.6 p < 0.0001] and feeding duration [F (2, 18) = 104.6 p < 0.0001] in satiated rats, whereas the latency to start feeding was decreased [F (2, 18) = 59.95 p < 0.0001] by the same dose (Table 1). To verify whether this finding is specific for injections centered in the DR, an additional group of rats received injections of CLO 20 nmol in the decussation of the superior cerebellar peduncle (xscp), a mesopontine area located between the DR and median

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raphe nucleus (MR). Findings in this group were compared to the intra-DR vehicle and CLO

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20 nmol groups. The one-way ANOVA revealed that CLO 20 nmol injection in the xscp did not affect the ingestive behaviors when compared to the intra-DR vehicle group

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(Supplementary Figure 1). Moreover, the intra-DR injection of CLO 20 nmol increased food

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intake [F (2, 16) = 21.19 p < 0.0001] when compared to the CLO 20 nmol injection into the xscp (Supplementary Figure 1). The amount of food consumed after CLO injections into DR

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in satiated animals represents the double compared to food intake observed in MR experiments using the same drug dose (Mansur et al., 2010). The nearby location of these

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two nuclei in the mesopontine tegmentum raises the possibility of diffusion of the drug from

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DR to MR, and the effects observed on food intake could be the result of the sum of the drug effect over these two nuclei. However, the lack of effect on food intake of CLO injections

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into the xscp almost excludes this possibility.

3.4

CLO injection into DR induces Fos expression in the PVN and ARC and this effect

is partially dependent on food intake To investigate the consequences of modifications in the adrenergic circuitry in the DR on neuronal activation profile in hypothalamic nuclei, immunohistochemical analyzes were performed in three different experimental groups, control subjects, rats with access to food (AF group) and rats without access to food (WAF group) during the experimental recording. For that, the 20 nmol dose of the α-2 adrenergic agonist clonidine was chosen, which caused a significant hyperphagic response. CLO 20 nmol injection into the DR in the AF group increased the number of Fos+ cells in the PVN when compared to the vehicle-AF group and CLO 20 nmol-WAF group [F (2, 12) = 6.93 p = 0.01]. However, the injection of clonidine in the WAF group failed to increase Fos expression in the PVN when compared to

the vehicle-AF group (Figure 2A-D), indicating that food intake is required to induce neuronal activity in PVN neurons. In the ARC, CLO 20 nmol injection into the DR in the AF group increased the number of Fos+ cells when compared to the vehicle-AF group and CLO 20 nmol-WAF group [F (2, 12) = 47.87 p < 0.0001]. However, the injection of clonidine into the DR in the WAF group also increased Fos expression in the ARC when compared to the vehicle-AF group (Figure 2E-H). Other brain nuclei analyzed, such as LHA, VMH, and BLA, did not show statistically significant changes in Fos expression between the experimental groups (Supplementary Table 1). 3.5

Food intake after CLO injection into DR causes Fos expression in POMC neurons,

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whereas non-POMC cells in the ARC are activated independently of food intake

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POMC neurons in the ARC induce satiety (Schwartz et al., 2000). Thus, we

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determined whether Fos expression was observed in POMC neurons by co-localizing Fos

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with alpha-melanocyte-stimulating hormone (α-MSH), which is a POMC-derived peptide. The mean number of α-MSH+ cells per section along the experimental groups analyzed was

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similar, suggesting that the sampling of these cells was not biased in any of the experimental conditions (Figure 3A). The treatment with CLO 20 nmol into the DR of AF group

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increased the percentage of Fos+ cells when compared to the vehicle-AF group and CLO 20

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nmol-WAF group [F (2, 12) = 5.81 p = 0.02] (Figures 3B and 3D). Double-labeled neurons were easily distinguishable by their α-MSH+ soma surrounding a Fos+ nucleus. However,

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the injection of clonidine in the WAF group failed to increase the percentage of doublelabeled cells compared to vehicle-AF group (Figures 3B and 3D). The number of Fos+ cells that did not co-localized with α-MSH increased with CLO 20 nmol injection in AF and WAF group when compared to the vehicle-AF group [F (2, 12) = 5.65 p < 0.02] (Figures 3C and 3D). The ARC is a hypothalamic nucleus that hosts different neuronal populations which are preferentially distributed in distinct subregions (Zhang and van den Pol, 2016). In this sense, POMC neurons are primarily located in the ventrolateral (VL) division of the ARC, whereas AgRP/NPY neurons are found in the ventromedial (VM) division. On the other hand, tyrosine hydroxylase (TH)-expressing neurons in the ARC are mainly located in the dorsomedial (DM) division. All these neuronal populations regulate feeding behaviors (Cone, 2005; Elmquist et al., 1999; Zhang and van den Pol, 2016). Thus, we performed an additional analysis to identify which ARC subdivisions were affected by CLO 20 nmol injection, independently of food intake, since an increased Fos expression was explained by

non-POMC cells. The injection of CLO 20 nmol in AF and WAF group increased the number of Fos+ cells in the VM division when compared to the vehicle-AF group [F (2, 12) = 9.95 p = 0.003] (Supplementary Figure 2B), while in the DM and VL divisions there was no statistical difference between the analyzed groups (Supplementary Figure 2C and 2D). 4

Discussion The results of the present study revealed that injections of NA or AD into DR

induced food intake in satiated rats. The behavioral response was characterized by increased duration and frequency of feeding and decreased latency to start feeding. The hyperphagia

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triggered by the NA or AD infusions into DR might be due to the activation of αadrenoceptors presents in the DR. Experiments with α-1 (PHE) and α-2 (CLO) specific

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adrenergic agonists support this hypothesis. CLO injection into DR of satiated rats caused

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hyperphagia similar to the response induced by NA or AD injection. The fact that CLO, but

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not PHE, induced hyperphagia, suggests that activation of α-2 pre-synaptic adrenoceptors has a stimulatory influence on feeding in this nucleus. In line with these data, previous

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studies revealed that AD injections into the median raphe nucleus (MR), another major serotonergic cell group displaying α-adrenoceptors (Adell and Artigas, 1999), increases food

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intake in satiated rats (dos Santos et al., 2009).

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There is manifold evidence that α-2 pre-synaptic adrenoceptors exert control over 5HT release in the DR. CLO injection into DR decreases 5-HT release, and lesion with DSP-

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4, a neurotoxin that impairs noradrenergic projections, abolishes CLO effects (Bortolozzi and Artigas, 2003). Thus, a decrease in neuronal activity, possibly serotonergic, is expected in the DR after injections of α-2 adrenergic agonists in satiated animals, leading to a decrease in 5-HT release in projection areas, which could favor ingestive behaviors. In line with this, inhibition of DR 5-HT neurons by local injection of 5-HT1A receptor agonist (8OH-DPAT) increased feeding in satiated rats (Hutson et al., 1986). In the present study, DR α-1 adrenergic activation failed to affect feeding behavior. One possible explanation is that satiated animals exhibit an elevated α-1 adrenergic tonus and, consequently, elevated inhibitory serotoninergic tone. In this case, PHE might not be able to bind to α-1 adrenoceptors since these receptors would already be occupied by endogenous NA released by noradrenergic terminals. In support of such a scenario, previous studies disclosed that PHE injections into MR also failed to affect ingestive behavior (Mansur et al., 2011a). Moreover, prazosin injections (α-1 adrenergic antagonist) increase food intake in satiated animals (Mansur et al., 2011b; da Silva et al., 2017), suggesting that in satiated rats the

activity of α-1 adrenoceptors in the MR is elevated. When MR α-2 adrenoceptors were blocked by yohimbine (α-2 adrenergic antagonist) followed by injection of AD, AD-induced hyperphagia was absent (Levone et al., 2015). Thus, we postulate that effect of NA or AD DR injections in satiated rats may be mediated through α-2 adrenoceptors, which acts as auto receptors that control endogenous NA release decreasing noradrenergic tonus in the DR, similar as it has been shown in the MR (Levone et al., 2015; Mansur et al., 2010; Mansur et al., 2011b). Furthermore, it is well established that serotonergic tonus is low in food restricted animals (Haider and Haleem, 2000) and the optical density of 5-HT positive neurons decreases significantly in the DR of rats submitted to food restriction when

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compared to satiated (Kang et al., 2001). However, it is imperative to carry out future experiments with adrenergic antagonists and with fasted animals in order to better elucidate

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the role of DR α-1 and α-2 adrenoceptors in this context.

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Although a substantial number of neurons in the DR are serotonergic, several other

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neuronal populations, such as GABAergic, dopaminergic, purely glutamatergic and neurons with a mixed glutamatergic/serotonergic phenotype, have been described in this nucleus

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(Hioki et al., 2010; Sego et al., 2014; Ren et al., 2019). A recent study disclosed that DR GABAergic and glutamatergic neurons are mutually activated by changes in energy balance

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and optogenetic and chemogenetic modulation of these neurons has opposite effects on food

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intake (Nectow, et al., 2017). In all, the findings of Nectow et al. (2017) indicate that, treatments that enhance the GABAergic tone within DR lead to an increase in food intake,

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an effect which may be mediated by local circuit mechanisms within DR. It is well known that DR GABAergic neurons inhibit serotonergic neurons, decreasing the release of 5-HT (Adell et al., 2002; Forchetti and Meek, 1981). The presence of adrenergic receptors in GABAergic and glutamatergic neurons in other brain regions, such as the basal forebrain (Holmberg et al., 1999; Midirrousta et al., 2004), and ventral tegmental area is well documented. Furthermore, a smaller percentage of DR GABAergic neurons express α-1b adrenoceptor mRNA (Day et al., 2004) and in a previous study, 5-HT, as well as non-5-HT neurons were activated in the DR, but not in the MR, by the ICV injection of AD 20 nmol (Flores et al., 2018). In all, we cannot completely rule out that non-serotonergic DR neurons participate in the effects caused by the injection of adrenergic agonists into the DR, but it seems unlikely that DR GABAergic neurons critically contribute to the responses observed due the absence of results after PHE injections into DR. Moreover, there is no evidence, until the present moment, that α-2 adrenoceptors are expressed in GABAergic or glutamatergic DR neurons.

Feeding frequency, feeding duration, and latency to start feeding have been linked to changes in mechanisms that initiate or end the meal (Blundell, 1986; Ritter and Epstein, 1975). The increase in frequency/duration and the decrease in feeding latency found after the adrenergic agonist injections into DR suggest that adrenergic mechanisms in the DR participate in the control of feeding initiation in this nucleus. Food intake, gastric distension, as well as cholecystokinin-induced satiety increase Fos protein expression in catecholaminergic neurons of the nucleus of the solitary tract (NTS) (Kim et al., 2020; Monnikes et al., 1997; Willing and Berthoud, 1997) and stimulate the release of catecholamines in projection areas (Buller and Day, 1996). Several studies have documented

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increased Fos-induction in A2/C2 catecholaminergic neuronal clusters in the brainstem in response to anorexic peptides such as cholecystokinin (Blevins et al., 2003; Rinaman et al.,

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1993). As previously mentioned, the DR does not display noradrenergic cell bodies, but

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receives robust NTS noradrenergic input (Peyron et al., 1996). Based on these findings, it is

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tempting to speculate that peripheral satiety signals might indirectly modulate the activity of DR neurons, inducing satiety via an increase in the activity of NTS noradrenergic neurons

neuronal network involved.

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that innervate the DR. However, further studies are required to better understand the

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CLO treatment induces Fos in ARC and PVN neurons in rats that have access to food

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during the experimental recording. However, when food is not offered during the experiment, PVN neurons fail to be activated, whereas, in the ARC, Fos-induction remains

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high compared to the control group. Together, these data suggest that ARC neurons could mediate hyperphagia induced by the CLO injection into the DR of satiated rats. After investigating the chemical phenotype of ARC activated neurons, we found Fos-induction in ARC POMC neurons and also in non-POMC neurons. ARC POMC neurons are largely related to inhibitory effects on feeding behavior (Schwartz et al., 2000) and produce α-MSH, an endogenous agonist of melanocortin 3 and 4 (MC-3/MC-4) receptors that is associated with anorexigenic effects (Cone, 2005; Elmquist et al., 1999; Williams and Schwartz, 2005). POMC neurons are known to express 5-HT2C receptors which play a critical role in regulating energy homeostasis (Heisler et al., 2003, Qiu et al., 2007; Xu et al., 2008). Moreover, 5-HT causes direct release of α-MSH in hypothalamic slices (Tiligada and Wilson, 1989). Considering this scenario, our expectation was to find fewer POMC neurons activated after CLO injection. However, the increased activity may be linked to the simple fact that the rats ingested food and not by a direct influence of the drug. Accordingly, in the WAF group POMC neurons exhibited a similar Fos expression pattern compared to vehicle-

injected animals. Corroborating these data, it has been shown that POMC neurons increase Fos expression after food intake (Fekete et al., 2012; Wu et al., 2014). The increased activity of ARC POMC neurons in the AF group can also explain Fos-induction in the PVN. PVN receives axonal input from ARC and expresses MC-3/MC-4 receptors (Atasoy et al., 2012; Cone et al., 2001). Local administration of MC-3/MC-4 agonists into PVN reduces food intake and intracerebroventricular injections of melanotan II, a mixed MC-3/MC-4 agonist induce PVN Fos expression (Giraudo et al., 1998; Rowland et al., 2010). Based on these data, food consumption during the experiment may have activated the ARC-PVN pathway. Our immunofluorescence analysis also revealed an increase in Fos-induction in non-

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POMC ARC neurons following CLO injection in rats without access to food during the experiment. Since we did not perform additional immunofluorescence analyses, we can only

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speculate, in which additional ARC neuronal populations Fos-induction occurred. Beside

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POMC, several other neurotransmitters and neuropeptides are expressed in the ARC

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(Meister, 2007; Campell et al., 2017; Zhang & van den Pol, 2016). The distinct location of Fos+/POMC- cells in the ARC may provide some hints. Thus, the fact that many of this

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Fos+/POMC- cells were found in the VM division of the ARC suggest that among them may be NPY/AgRP neurons, which are mostly restricted to this subdivision. NPY/AgRP neurons

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are potential candidates to mediate the hyperphagia induced by the CLO injection because

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they have well-known orexigenic effects on food intake (Aponte et al., 2011; Zhang et al., 2019). Recently, a third population of neurons located in the ARC that express tyrosine

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hydroxylase (TH) has been described to be confined to the DM and VL divisions of the ARC and characterized by its orexigenic effects on food intake (Zhang and van den Pol, 2016). However, since DM and VL ARC subdivisions did not exhibit substantial Fosinduction after CLO injections, the participation of ARC TH neurons in the feeding response evoked by DR is unlikely. In conclusion, the data from the present study highlight that the pharmacological activation of DR α-adrenoceptors increases food intake in satiated rats. The feeding response evoked by injections of the α-2 agonist CLO into DR was similar to that induced by NA or AD injections, suggesting that the hyperphagia observed after NA or AD treatment depends on α-2 adrenoceptors activation. Moreover, the stimulation of α-1 adrenergic receptors within the DR had no impact on feeding behavior in satiated rats. Finally, CLO injections into DR increased neuronal activity in the ARC, possibly evoking a homeostatic response toward food intake. Although our findings not necessarily reflect a physiological mechanism, since rats are mostly nocturnal feeders, whereas our feeding experiments

occurred in the morning, they indicate a potential role of noradrenergic and serotonergic mechanisms in food intake that warrants further investigation. Declaration of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author contributions RF and MP conceived the study and designed experiments. RF performed all the experiments with the participation of RS in the behavioral tests. RF, JP and JD performed

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immunohistochemical and immunofluorescence analyzes, and JD supplied reagents and materials. MM contributed to the image collection and data analysis. RF wrote the

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manuscript with input from all other authors and revision from MP, MM, and JD.

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Acknowledgments

Paulo

Research

Foundation

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This study was funded by Brazilian Governmental agencies, CNPq, CAPES, and São (FAPESP-Brazil,

2017/02983-2,

2012/15517-6,

and

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2017/16473-6). We would like to thank Ana Maria Peracoli Campos for the technical support provided with immunohistochemical procedures, and Multiuser Laboratory of

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Studies in Biology (LAMEB/UFSC) staff for the technical assistance.

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Figure 1: Confirmation of the injection sites and food intake after injection of adrenergic agonists into DR of satiated rats. A) Photomicrograph of a stained section, showing the injection into the DR. B) Injection sites at the bregma level: -7,80 mm; other injection sites were located at -7.32 to -8.04 mm to bregma. Abbreviations: Aq - aqueduct; DR - dorsal raphe nucleus; DMPAG - dorsomedial periaqueductal gray; LPAG - lateral periaqueductal gray; VLPAG – ventrolateral periaqueductal gray; mlf - medial longitudinal fasciculus. C) Changes in the amount of food intake after injection with vehicle (VEH) or adrenaline (AD) at 6 nmol, 20 nmol, and 60 nmol doses into the DR of satiated rats. D) Changes in the amount of food intake after treatment with vehicle (VEH) or noradrenaline (NA) at 6 nmol, 20 nmol, and 60 nmol doses into the DR of satiated rats. E) Changes in the amount of food intake after injection with vehicle (VEH) or the α-1 adrenoceptor agonist phenylephrine (PHE) at 6 nmol and 20 nmol doses into the DR of satiated rats. F) Changes in the amount of food intake after injection with vehicle (VEH) or the α-2 adrenoceptor agonist clonidine (CLO) at 6 nmol, 20 nmol doses into the DR of satiated rats. In all sets of experiments, separate animals were used across doses, in which each animal received a single injection of drug or vehicle. Data represent the mean ± SEM * p < 0.05 vs vehicle group. Non-paired one-way ANOVA followed by Tukey's post hoc test (n = 6 - 8 per group).

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Figure 2: Fos expression in the paraventricular nucleus (PVN) and in the arcuate nucleus (ARC) after injection of CLO 20 nmol into the DR of satiated rats. CLO 20 nmol were injected into DR of new experimentally naive satiated rats with access to food during the experiment (access to food group – AF) or that have no access to food during the behavioral recording (without access to food group –WAF). A) Graphical illustration displaying the average number of Fos + cells per section in the PVN of AF and WAF groups. Data represent the mean ± SEM. * p < 0.05 CLO20 nmol-AF group vs vehicle-AF group. ** p < 0.05 CLO 20 nmol-AF group vs CLO 20 nmol-WAF group. Non-paired one-way ANOVA followed by Tukey’s post hoc test (n = 5 per group). VEH = vehicle; CLO = clonidine; NS = non-significant; AF = Access to food group; WAF = Without access to food group. B) Fos expression in a representative section of the PVN in rats of the vehicleAF group. C) Fos expression in a representative section of the PVN in rats of the CLO 2O nmol-AF group. D) Fos expression in a representative section of the PVN in rats of the CLO 2O nmol-WAF group. 3V: third ventricle; Scale bar = 100 µm. E) Graphical illustration displaying the average number of Fos+ cells per section in the ARC of AF and WAF groups. Data represent the mean ± SEM. * p < 0.05 CLO20 nmol-AF group vs vehicle-AF group. ** p < 0.05 CLO 20 nmol-AF group vs CLO 20 nmol-WAF group. *** p < 0.05 CLO 20 nmol-WAF group vs vehicle-AF group. Non-paired one-way ANOVA followed by Tukey’s post hoc test (n = 5 per group). VEH = vehicle; CLO = clonidine; AF = Access to food group; WAF = Without access to food group. F) Fos expression in a representative section of the ARC in rats of the vehicle-AF group. G) Fos expression in a representative section of the ARC in rats of the CLO 2O nmol-AF group. H) Fos expression in a representative section of the ARC in rats of the CLO 2O nmol-WAF group. 3v = third ventricle; VMH = ventromedial hypothalamus. Scale bar = 100 µm. Figure 3: Fos expression and its co-localization with POMC neurons (α-MSH+/ Fos+) in the ARC. CLO 20 nmol were injected into DR of new experimentally naive satiated rats with access to food during the experiment (access to food group – AF) or that have no access to food during the behavioral recording (without access to food group –WAF). A) Total number of α-MSH+ cells in the ARC after CLO 20 nmol injection into the DRN of AF and WAF groups. B) Percentage of α-MSH+/ Fos+ cells in the arcuate nucleus (ARC) after CLO 20 nmol injection into DR of AF and WAF groups. Data represent the mean ± SEM. * p < 0.05 CLO20 nmol-AF group vs vehicle-AF group. ** p < 0.05 CLO 20 nmol-AF group vs CLO 20 nmol-WAF group. Nonpaired one-way ANOVA followed by Tukey’s post hoc test (n = 5 per group). C) The number of cells only Fos+ in the ARC after CLO 20 nmol injection into DR of AF and WAF groups. Data represent the mean ± SEM. * p < 0.05 CLO20 nmol-AF group vs vehicle-AF group. ** p < 0.05 CLO 20 nmol-WAF group vs vehicle-AF group. Non-paired one-way ANOVA followed by Tukey’s post hoc test (n = 5 per group). VEH = vehicle; CLO = clonidine; NS = non-significant; AF = Access to food group; WAF = Without access to food group. D) Immunofluorescence photomicrographs illustrating the co-localization of the Fos protein in α-MSH + neurons located in the ARC. In red, α-MSH + cells and green, Fos + cells. In the merged images, the white arrows represent examples of double labeled neurons. AF = Access to food group; WAF = Without access to food group; alpha-MSH = alpha-melanocyte-stimulating hormone; ARC = arcuate nucleus; 3v = third ventricle. Scale bar = 25 µm.

Supplementary legends Supplementary Figure 1: Effect of the clonidine 20 nmol injection into the decussation of the superior cerebellar peduncle (xscp) on food intake in satiated rats. Data represent the mean ± SEM * p < 0.05 CLO 20 nmol-DR group vs vehicle-DRN group. ** p < 0.05 CLO 20 nmol-DR group vs CLO 20 nmol xscp group. Non-paired one-way ANOVA followed by Tukey's post hoc test. VEH = vehicle; CLO = clonidine; MR = median raphe nucleus; Aq = cerebral aqueduct; DR = dorsal raphe nucleus; NS = non-significant.

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Supplementary Figure 2: Fos expression in the different subdivisions of the arcuate nucleus (ARC). A) A schematic of divisions of the ARC. VM = ventromedial; VL = ventrolateral; DM = dorsomedial; ME = median eminence; 3v = third ventricle. B) The number of cells only Fos+ in the ventromedial division of ARC after CLO 20 nmol injection into DR of AF and WAF groups. Data represent the mean ± SEM. * p < 0.05 CLO20 nmol-AF group vs vehicle-AF group. ** p < 0.05 CLO 20 nmol-WAF group vs vehicle-AF group. Non- paired one-way ANOVA followed by Tukey’s post hoc test (n = 5 per group). C) The number of cells only Fos+ in the dorsomedial division of ARC after CLO 20 nmol injection into DR of AF and WAF groups. Data represent the mean ± SEM. One-way ANOVA (n = 5 per group). D) The number of cells only Fos+ in the ventrolateral division of ARC after CLO 20 nmol injection into DR of AF and WAF groups. Data represent the mean ± SEM. Non-paired one-way ANOVA (n = 5 per group). VEH = vehicle; CLO = clonidine; NS = nonsignificant; AF = Access to food group; WAF = Without access to food group.

Highlights: Dorsal raphe activation of α-adrenoceptors increases food intake in rats. Dorsal raphe injections of noradrenaline increase food intake in fed rats. Dorsal raphe injections of α-2 agonist clonidine increase food intake in fed rats. Dorsal raphe injections of α-1 agonist phenylephrine do not change feeding behavior.

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Dorsal raphe injection of clonidine increases Fos expression in the arcuate nucleus.