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Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats
PBB-71986; No of Pages 6 Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
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Eduardo Simão da Silva ⁎, Rafael Appel Flores, Elisa Carolina Cella, Brunno Rocha Levone, Ana Paula Taschetto, Larissa Kochenborger, Mariana Graciela Terenzi, Moacir Serralvo Faria, Marta Aparecida Paschoalini
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Department of Physiological Sciences, Center of Biological Sciences — CCB, Federal University of Santa Catarina (UFSC), 88040-970, Florianópolis, SC, Brazil
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Article history: Received 29 March 2014 Received in revised form 11 June 2014 Accepted 15 June 2014 Available online xxxx
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Keywords: Median raphe nucleus α1-Adrenoceptor Food intake Rat
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Previous studies have shown that the blockade of α1-adrenoceptors in the median raphe nucleus (MnR) of free-feeding animals increases food intake. Since there is evidence for the presence of α1A-, α1B- and α1Dadrenoceptors in the MnR of rats, this study investigated the involvement of MnR α1-adrenoceptor subtypes in the control of feeding behavior, looking for possible differences on the role of each α1-adrenoceptor in feeding. Male adult rats weighing 280–300 g with guide cannulae chronically implanted above the MnR were injected with antagonists of α1A- (RS100329, 0, 2, 4 or 20 nmol), α1B- (Rec 15/2615, 0, 2, 4 or 20 nmol) or α1Dadrenoceptor (BMY 7378, 0, 2, 4 or 20 nmol). Subsequently, behavioral evaluation of ingestive and noningestive parameters was monitored for 1 h and the amount of food and water ingested was assessed for 4 h. The highest dose (20 nmol) of RS100329 and BMY 7378 increased food intake, feeding duration and frequency, and decreased the latency to start feeding. During the second hour 2 nmol dose of Rec 15/2615 increased food intake and all doses of BMY 7378 decreased water intake. No behavioral alterations were observed during the fourth hour. The results corroborate previous work from our lab in which we describe the involvement of α1-adrenoceptors of MnR on food intake control. Moreover, we show evidence that α1A- and α1D-adrenoceptors mediate feeding responses to adrenaline injections and that the behavioral modifications are of considerable duration, persisting up to 2 h after injection of the antagonists. © 2014 Published by Elsevier Inc.
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1. Introduction
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The raphe nuclei are distributed rostro-caudally throughout the brainstem and present distinct neurochemical, morphological and projection characteristics (Wirtshafter, 2001; Adell et al., 2002; Hornung, 2003; Walther and Bader, 2003; Mokler et al., 2009; Takase and Nogueira, 2008). The median raphe nucleus (MnR) and the dorsal raphe nucleus send numerous serotonergic fibers to prosencephalic structures (Lucki, 1998; Hornung, 2003; Mokler et al., 2009). While the dorsal raphe nucleus innervates mainly the amygdala, ventral hippocampus and striatum (Azmitia and Segal, 1978), the MnR innervates preferentially the dorsal hippocampus, medial septum, nucleus accumbens, ventral tegmental area and several hypothalamic nuclei (Vertes et al., 1999; Lechin et al., 2006). The MnR has a diversity of neurotransmitter systems involved in the control of food intake, it is rich in 5-HT1A receptors (Sotelo et al., 1990; Kia et al., 1996; Cryan et al., 2002; Adell et al., 2002; Judge and Gartside, 2006) and the major part of these receptors is found in
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Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats
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⁎ Corresponding author. Tel.: +55 48 37219352; fax: +55 48 37219672. E-mail address: [email protected] (E.S. da Silva).
serotonergic neurons acting as autoreceptors (Hall et al., 1997; Adell et al., 2002). Injection of 8-OH-DPAT (a 5-HT1A antagonist) into the MnR inhibits serotonergic neuronal firing, 5HT release at prosencephalic structures (Bonvento et al., 1992; Andrews et al., 1994; Avanzi and Brandão, 2001; Adell et al., 2002; Funk et al., 2005) and increases food intake (Currie et al., 1994). Along with 5HT receptors, GABA and glutamatergic receptors localized in the MnR also appear to participate in food intake regulation. Intra-MnR injection of kainic or quisqualic acid suppresses food intake in food-deprived rats, on the other hand antagonists of kainate/quisqualate receptors increase food intake in free-feeding rats (Wirtshafter and Krebs, 1990). Intra-MnR injection of Baclofen, a GABAB agonist, increases food and water intake and induces locomotor activity in non-deprived (free-feeding) rats (Wirtshafter et al., 1993). Data from our lab have shown that adrenaline (AD) injected into the MnR increases food intake in free-feeding rats (Maidel et al., 2007), but reduces food intake in food-deprived rats (Dos Santos et al., 2009). These opposite effects of AD were attributed to differential activation of pre-synaptic and post-synaptic α-adrenoceptors that is dependent on the nutritional state of the animal. Later data showed that MnR injections of prazosin (Mansur et al., 2011), a α1-adrenoceptor antagonist, and clonidine, a α2-adrenoceptor agonist, increased food intake in
http://dx.doi.org/10.1016/j.pbb.2014.06.010 0091-3057/© 2014 Published by Elsevier Inc.
Please cite this article as: da Silva ES, et al, Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.010
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2.1. Animals
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Male Wistar rats (weighing 270–300 g at the time of surgery) were group-housed at 22–24 °C, 12:12 light–dark cycle (lights on at 6:00 AM) with standard rodent chow and water available ad libitum. The animals were housed in groups of five per cage until the day of the experiments. After surgery, rats were housed in individual cages. The experimental procedures were conducted in compliance with the recommendations of the Ethics Committee for the use of Experimental Animals (CEUA) of the Federal University of Santa Catarina, SC, Brazil (CEUA protocol: #PP0075-2010). All efforts were made to minimize the number of animals used and their suffering.
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The rats were anesthetized (ip) with a ketamine hydrochloride (87 mg kg−1) and xylazine (13 mg kg−1) mixture and stereotaxically implanted with a unilateral stainless steel guide cannula (30G, 18 mm length). The target for this cannula was 2 mm above the MnR based on the atlas of The Rat Brain (Paxinos and Watson, 2007). The following coordinates from bregma were used: AP = −7.8 mm; L = 3.0 mm; and DV = 7.0 mm from the surface of the skull, at an angle of 20° from the vertical plane to avoid the sagittal sinus and the cerebral aqueduct. The cannula was anchored to the skull with dental cement and the whole implant stabilized with jeweler screws and more dental cement. A removable stylet was introduced to keep the cannula free from blockages until the day of the experiment.
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2.4.1. Behavioral assessment After they have been injected, the animals were placed for 60 min in a cage containing a known weight of standard rodent chow and a known volume of faucet water. At the end of the session, the measured difference between food and water at the beginning and at the end was taken as the amount of food or water consumed. The experiment was recorded with a webcam for subsequent behavioral analysis with the Etholog 2.25 (Ottoni, 2000). During the session the duration (time spent in a given behavior during experimental time), frequency (number of episodes of a given behavior) and latencies (time in seconds to initiate a given behavior) for eating, drinking, locomotion, sniffing, immobility, rearing and grooming were evaluated. See Ribas et al. (2012) for details about these procedures and description of behaviors. In addition, food and water intake recordings were also taken 2 and 4 h post-injection.
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2.5. Histological analysis
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At the end of the experiments, the animals were deeply anesthetized and transcardially perfused with saline (0.9%) and formalin (10%). The brains were removed, kept in formalin and sliced in the coronal plane (50 μm). Sections were mounted on gelatinized slides and stained with cresyl violet. The cannula placements were identified under a microscope by comparison of the sections with photographs and diagrams of The Rat Brain atlas (Paxinos and Watson, 2007). Only data from rats with cannula correctly placed in the MnR were included in the study (approximately 80% of the total of implanted animals).
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2.6. Statistical analysis
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Behavioral data were analyzed by one-way ANOVA followed by Duncan's post hoc. The amount of food and water consumed during the experiment was analyzed by repeated measures ANOVA. Results are expressed as mean ± standard error of the mean (SEM). When appropriate, the ANOVA was followed by Duncan's post hoc test for multiple comparisons. Only probability values of less than 5% were considered significant.
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3. Results
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2.3. Drugs and injections
3.1. α1A-Adrenoceptor antagonist
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Injections were made using a needle (33G, 20 mm length) extending 2 mm beyond the ventral tip of the guide cannula and connected by polyethylene tubing (PE10) to a Hamilton microsyringe (1 μl) attached to an injection pump. The injected volumes (0.2 μl) were administered over a period of 60 s and a further 60 s was allowed for the solution to diffuse from the needle. RS100329 a α1A-adrenoceptor antagonist, Rec15/2615 a α1B-adrenoceptor antagonist, and BMY7378 a α1Dadrenoceptor antagonist were given at doses of 2, 4 and 20 nmol respectively, purchased from TOCRIS Biosciences (Ellisville, MO, USA) and dissolved in 0.9% sterile saline with 5% DMSO. Each animal received only one injection: a dose of one drug or the corresponding vehicle.
All animals included in the statistical analysis had their injection site verified by histological assessment. The ANOVA of repeated measures revealed that a 20 nmol dose of RS100329 (Fig. 1) significantly increased food intake in the first hour (F(6, 68) = 3.52; p = 0.03). This treatment increased both duration (F(3, 34) = 6.5429; p = 0.001) and frequency of feeding (F(3, 34) = 7.49; p = 0.0005); in addition, a decreased latency (F(3, 34) = 4.1198; p = 0.01) to start eating was observed (Table 1). The 20 nmol dose of RS100329 did not modify drinking or non-ingestive behaviors. Both lower doses of RS100329 produced no effect on any behavior registered in this study (Table 2). Water intake was not significantly altered at any interval studied.
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Animals were acclimatized to the feeding recording chamber for 1 h for two consecutive days immediately before the experimental session. The experiment was designed to evaluate the effects of RS100329 (n = 10 for each dose), Rec 15/2615 (n = 10 for each dose) or BMY7378 (n = 9 for each dose) injection (0, 2, 4 or 20 nmol) into the MnR on ingestive and non-ingestive behaviors during the first hour. The ingestive behavior was also evaluated 2 and 4 h after drug injection.
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free-feeding rats (Mansur et al., 2010), supporting the hypothesis of a differential activation by AD. On the other hand, MnR injections of phenylephrine, a α1-adrenoceptor agonist, decreased food intake in food-deprived rats while clonidine had no effect (Ribas et al., 2012). Collectively, these results allowed us to conclude that in free-feeding animals, the activation of MnR α1-adrenoceptor receptors stimulates an inhibitory pathway on food intake. The activity of this inhibitory pathway appears to be reduced in food-deprived conditions. MnR neurons receive afferents from the locus coeruleus, lateral tegmental area, A1/A2 cell group and AD C1/C2 medullary nuclei (Hopwood and Stamford, 2001; Cryan et al., 2002; Adell et al., 2002; Lechin et al., 2006) and express mRNA of α1-adrenoceptor subtypes α1A, α1B and α1D (Day et al., 1997). There is evidence that a noradrenergic input to the MnR exerts tonic facilitatory control of 5-HT release through α1-adrenoceptors (Adell and Artigas, 1999). Given the presence of α1-adrenoceptor subtypes in the MnR, the aims of our work are to study the effect of various α1-adrenoceptor antagonists injected into the MnR on feeding behavior and to determine the duration of such effects by prolonging the period of observation of food and water intake used in previous studies.
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Please cite this article as: da Silva ES, et al, Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.010
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Fig. 1. Food intake pattern after MnR injection of vehicle (white bars), 2 (light gray bars), 4 (dark gray bars) or 20 (black bars) nmol of RS100329, Rec15/2615 or BMY 7871 in free-feeding rats. Values are mean ± SEM. *p ≤ 0.05 compared to vehicle treatment (repeated measures ANOVA followed by Duncan's post hoc test).
3.3. α1D-Adrenoceptor antagonist
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The 20 nmol dose of BMY7378 (Fig. 1) significantly increased food in202 take in the first hour (F(6, 56) = 6.17; p = 0.003) and evoked significant 203 changes in this behavior increasing both duration (F(3, 29) = 9.23; 204 p = 0.0001) and frequency (F(3, 29) = 3.6, p = 0.02) and decreasing 205 latency (F(3, 29) = 6.9, p = 0.001) to start eating (Table 1). Non206 ingestive behaviors and drinking were not altered during the first hour. 207 All doses of α1D-adrenoceptor antagonist significantly decreased 208 water intake on the second hour after injection (Fig. 2; F(6, 56) = 209 Q10 9.17; p = 0.0003). No significant alterations in food or water intake 210 were observed on the fourth hour. BMY7378 in 2 or 4 nmol dose pro211 duced no effect on other behaviors registered in this study. 4. Discussion
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The present study shows that blockade of α1A- and α1Dadrenoceptors increases food intake approximately 28 min after drug injection. This response is not due to increased inespecific motor activity since the duration and frequency of non-ingestive behaviors remained unchanged. The behavioral response was characterized by increased duration and frequency and decreased latency to start feeding. Previous work showed that an α1-adrenoceptor antagonist, prazosin, injected in the MnR produced similar results (Mansur et al., 2011). Our work suggests that the prazosin effect observed in free-feeding rats was produced by blockade of α1A- and α1D-adrenoceptors. Conversely, the activation of α1-adrenoceptor in food-deprived rats resulted in a decrease in the feeding response after re-feeding (Ribas et al., 2012). Furthermore, the activation of α2-adrenoceptors results in hyperphagia in free-feeding animals (Mansur et al., 2010). This result could be attributed to an inhibition of AD release from presynaptic stores, thus removing the α1-adrenergic stimulatory tone on serotonergic neurons within the
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Interestingly, in the first hour of the experimental recording, the injection of a 4 nmol dose of Rec 15/2615, an α1B-adrenoceptor antagonist, significantly increased feeding duration (F(3, 36) = 5. 05; p = 0.005) (Table 1). In the same time interval, food intake remained unchanged as well as the frequency and the latency to start eating. During the second hour after drug injection, the 2 nmol dose of Rec 15/2615 significantly increased food intake alone (Fig. 1, F(6, 80) = 10.2, p = 0,0001), all other parameters of feeding behavior remained unchanged. No significant difference in feeding behavior was observed on the fourth hour of experimental session at any dose. Drinking and non-ingestive behaviors were not affected by drug treatments during the experimental period.
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MnR, since α2-adrenoceptor seems to exert a tonic inhibitory influence on 5-HT release in the MnR (Adell and Artigas, 1999). Taken together these data suggest that the adrenergic receptors within MnR stimulate a tonically inhibitory influence that restrains food intake under freefeeding conditions; the intensity of this inhibitory mechanism seems to decline as food availability decreases. One important issue to address related to feeding effects mediated by α1D-adrenoceptors, is that BMY 7378 is also a 5HT1A partial agonist. Thus, the increase in feeding observed in our experiments could be due to BMY 7378 acting through serotonergic receptors since it has been reported that 5HT1A activation within the MnR resulted in hyperphagia (Currie and Coscina, 1993). In fact, Higgins and Elliot (1991) showed that 8OH-DPAT, a 5HT1A agonist, as well as BMY 7378 injections into the MnR increased locomotion. However, the dose of BMY 7378 used to produce effects similar to those caused by 8-OH-DPAT was higher than the one used in our study. Thus, the dose herein used may not be high enough to activate 5HT1A receptors therefore leading to the suggestion that the feeding effects are due to activation of α1D-adrenoceptors. The extended period of observation of 2 and 4 h in our experiments revealed that α1B-adrenoceptors also have a delayed role in the control of food intake. In addition to the first hour of hyperphagia observed with α1A- and α1D-adrenoceptor antagonist administration, we also identified a late increase in food intake induced by α1B-adrenoceptor blockade. Although delayed and observed only with the lowest dose, the relevance of this mechanism could be indicated by the magnitude of the feeding response, similar to that evoked by prazosin or activation of α1A- and α1D-adrenoceptor subtypes. If this experimental design had been used in previous work, the feeding response evoked by α1Badrenoceptor might also have been seen after prazosin treatment. A possible explanation for the delayed feeding response of α1Badrenoceptor blockade is that, unlike α1A- and α1D-adrenoceptors, the signaling pathway that is being inhibited by Rec 15/2615 is a protein Gq coupled to phospholipase C. While the α1A- and α1D-adrenoceptors directly open Ca2 + channels in the membrane, α1B-adrenoceptors promote the formation of diacylglycerol and phosphatidylinositol 3-phosphate that, in turn, opens Ca2 + channels. Thus, blockade of α1B-adrenoceptors interrupts the trigger of an ongoing cascade that takes time to fully develop. However, further investigation is necessary to verify that assertion. Rec 15/2615 increased the duration of feeding for 1 h, without food intake (Table 1). Feeding frequency and the latency to start feeding were not affected by the blockade of α1B-adrenoceptors. The increase in feeding duration may be related to an intensification of oral exploration, or, on the other hand, oral motor activity could be affected by the treatment. In this situation, the animals could spend more time with food in the mouth before swallowing it, thus the prolonged duration of each eating episode significantly increased feeding duration. Previous studies have shown that rats spent more time chewing wood blocks
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Please cite this article as: da Silva ES, et al, Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.010
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t1:3
RS100329
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12
t2:1 t2:2
Feeding
Drinking
Duration Latency Frequency Duration Latency Frequency
2 nmol
34 ± 24 2982 ± 415 0.6 ± 0.4 0 3600 0
32 3281 0.5 34 3420 0.6
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4 nmol
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32 318 0.5 34 179 0.6
9 3319 0.3 7 3434 0.2
20 nmol
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227 1741 3.2 34 2842 0.6
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BMY 7871
0 nmol 67⁎ 421⁎ 0.7⁎ 21 350 0.2
34 2982 0.6 11 3385 0.8
± ± ± ± ± ±
24 415 0.4 11 214 0.8
2 nmol
4 nmol
0 3600 0 20 ± 19 3148 ± 304 0.6 ± 0.5
133 1672 2.6 80 2491 2
20 nmol
± ± ± ± ± ±
35⁎ 425 1.1 32 375 0.7
46 3225 1.1 62 2696 1.4
± ± ± ± ± ±
26 232 0.6 28 381 0.6
0 nmol
2 nmol
0 3600 0 3.1 ± 3.1 3264 ± 335 0.1 ± 0.1
26 2471 1.1 1.5 3273 0.1
± ± ± ± ± ±
4 nmol 11 435 0.3 1.5 326 0.1
226 1794 3.2 38 2150 1.5
± ± ± ± ± ±
20 nmol 77 429 1.3 19 556 0.6
353 1723 2.7 0.3 3315 0.1
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Table 2 Effects of intra-MnR injection of RS100329, Rec15/2615 or BMY 7871 on non-ingestive behaviors in free-feeding rats. RS100329
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Values are mean ± SEM. Duration and latency are expressed in seconds and frequency is expressed as number of events. ⁎ p ≤ 0.05 compared to vehicle treatment (one-way ANOVA followed by Duncan's post hoc test).
t2:3
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Table 1 Effects of intra-MnR injection of RS100329, Rec15/2615 or BMY 7871 on ingestive behaviors in free-feeding rats.
0 nmol Sniffing
Immobility
Rearing
Grooming
Locomotion
Duration Latency Frequency Duration Latency Frequency Duration Latency Frequency Duration Latency Frequency Duration Latency Frequency
270 4 38 2888 78 22 45 29 12 325 20 24 55 4 25
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Rec 15/2615 2 nmol
35 1 3 59 25 3 6 10 1 23 6 2 5 2 2
485 5 54 2538 107 26 81 342 14 340 691 14 91 0 33
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4 nmol 92 1 10 182 38 3 28 269 4 70 450 4 15 0 6
464 2 54 2542 242 32 80 100 21 421 422 20 75 1 27
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
20 nmol 73 1 4 135 123 5 15 83 3 86 272 4 11 0 3
507 2 57 2032 84 27 73 288 24 479 93 17 100 1 39
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0 nmol 87⁎ 1 12 278 40 2 49 197 8 79 60 2 30 0 12
345 4 47 2736 88 27 55 18 13 339 24 24 69 4.1 31
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2 nmol 82 1 10 139 26 5 12 3 3 29 7 2 14 1.9 5
Values are mean ± SEM. Duration and latency are expressed in seconds and frequency is expressed as number of events. ⁎ p ≤ 0.05 compared to vehicle treatment (one-way ANOVA followed by Duncan's post hoc test).
716 2 65 2241 121 27 59 21 17 437 56 29 127 0.7 50
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
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552 1.8 64 2207 125 24 111 8 24 510 14 33 110 0.7 43
4 nmol 84 0.3 13 160 60 4 10 6 2 81 13 5 30 0.5 9
508 2 55 2095 441 25 69 31 28 425 51 33 110 0.5 40
138 0.4 9 128 242 5 13 10 6 76 12 7 21 0.3 6
BMY 7871
R O ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
89 0.4 11 147 24 3 28 3 5 98 8 8 20 0.3 7
0 nmol 537 2.7 112 2381 57 37 228 147 52 320 67 15 128 0.1 78
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
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2 nmol 97 0.6 23 219 21 4 58 135 15 62 11 3 34 0.1 18
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513 1.9 92 2344 135 29 256 78 45 358 121 11 112 0.4 60
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4 nmol 82 0.3 18 243 31 6 85 68 13 83 61 2 20 0.4 10
628 2.6 91 2071 90 29 162 24 31 379 78 15 119 1.3 63
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
20 nmol 107 0.8 13 167 28 2 54 7 7 67 24 2 24 1.3 12
736 1.6 108 1909 127 33 201 29 43 369 86 15 175 0.9 73
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
104 0.4 13 174 34 3 40 18 8 70 27 3 28 0.9 10
E.S. da Silva et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
Please cite this article as: da Silva ES, et al, Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.010
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E.S. da Silva et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
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Fig. 2. Water intake pattern after MnR injection of vehicle (white bars), 2 (light gray bars), 4 (dark gray bars) or 20 (black bars) nmol of RS100329, Rec15/2615 or BMY 7871 in free-feeding rats. Values are mean ± SEM. *p ≤ 0.05 compared to vehicle treatment (repeated measures ANOVA followed by Duncan's post hoc test).
after injection of 8-OH DPAT (Dourish et al., 1985) and muscimol (Klitenick and Wirtshafter, 1989) into the MnR. Klitenick and Wirtshafter (1989) attribute these results to an “energizing” effect of 280 oral behaviors. The authors claim that this result appears to be consis281 tent with the idea that MnR is an integral part of a system of non282 specific behavioral activation. We suggest that adrenergic receptors 283 could also be part of this circuit, however we did not observe statistical284 ly significant changes in the duration or frequency of behaviors that in285 dicate the intensity of motor activity (vertical exploration, locomotion, 286 grooming). Thus, we conclude that the increase in eating behavior 287 may be due to specific intensified oral behavior responses. 288 Water intake was significantly decreased 2 h after α1D-adrenoceptor 289 blockade. In contrast, previous results from our lab showed that MnR 290 injections of 20 nmol AD decreased the latency to start drinking (dos 291 Santos et al., 2009). Taken together these suggest that the circuit in 292 which AD receptors take part could also control water intake. However, 293 phenylephrine or clonidine evoked no change in water intake in 294 food-deprived rats at least for 30 min after drug injections (Ribas et al., 295 2012). Several studies have shown that serotonergic and non296 serotonergic systems participate in the control of food and water intake 297 (Wirtshafter, 2001). The participation of GABAA and GABAB receptors 298 within the MnR were revealed by increases seen in food and water intake 299 induced by the injection of GABA agonists, muscimol and baclofen 300 (Klitenick and Wirtshafter, 1988; Wirtshafter et al., 1993). In addition, 301 intra-MnR treatment with glutamatergic antagonists increased feeding 302 and drinking (Wirtshafter and Krebs, 1990; Wirtshafter and Trifunovic, 303 1988). Meanwhile, 5HT1A activation by 8OH-DPAT injected into the 304 MnR appears to cause alteration in food rather than water intake 305 (Currie and Coscina, 1993; Currie et al., 1994; Fletcher, 1991; Fletcher 306 and Coscina, 1993). In that way, the AD circuit within the MnR may be 307 interacting with 5HT, GABA or glutamate neurons to control mainly 308 food intake, while the participation of 5HT neurons as mediators of the 309 AD effects on water intake could be considered minor. 310 The effects of α-adrenoceptor antagonists on ingestive behavior 311 observed in our study are confined to the MnR since previous work 312 showed that the effects of non-specific agonist and antagonists are in313 duced only with injections placed within the MnR but not in the vicinity 314 (Ribas et al., 2012; Mansur et al., 2011). 315 Intriguingly, instead of a feeding response less intense than that 316 evoked by prazosin, a full antagonist of α-adrenoceptors, the blockade 317 of individual α-adrenoceptor subtypes resulted in a feeding response 318 increase that was similar to that induced by prazosin. This may be ex319 plained by the fact that all three receptor subtypes' signaling converges 2+ 320 Q12 onto similar modifications of Ca concentrations. 321 The present work shows the involvement of α1A- and α1D322 adrenoceptors present in the MnR in the rapid control of food intake. 323 It also demonstrates a delayed control on food intake by the blockade of 324 α1B-adrenoceptor that resulted in hyperphagia. We further demonstrate
Acknowledgments
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We would like to thank the technical staff from LAMEB2, Mrs. Chirle Ferreira, Mr. Demétrio Gomes Alves and Ms. Emily Daiana dos Santos for their technical assistance. This study was supported by CNPq research grant to M. A. Paschoalini and research fellowship to E. S. Silva.
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the participation of AD receptors of MnR on water intake by showing that α1D-adrenoceptor blockade caused a reduction in water intake. Although the treatment with specific antagonists of α-adrenoceptors reproduced previous findings to those of a full antagonist, individual receptor blockade was justified by the variety of responses encountered on delayed feeding and water responses.
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Mansur SS, Terenzi MG, Marino-Neto J, Faria S, Paschoalini MA. Alpha1 receptor antagonist in the median raphe nucleus evoked hyperphagia in free-feeding rats. Appetite 2011;57(2):498–503. Mokler DJ, Dugal JR, Hoffman JM, Morgane PJ. Functional interrelations between nucleus raphé dorsalis and nucleus raphé medianus: a dual probe microdialysis study of glutamate-stimulated serotonin release. Brain Res Bull 2009;78: 132–8. Ottoni EB. EthoLog 2.2: a tool for the transcription and timing of behavior observation sessions. Behav Res Methods Instrum Comput 2000;32:446–9. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 6th ed. NewYork: Academic Press & Elsevier Inc.; 2007. Ribas AS, Flores RA, de Nazareth AM, Faria MS, Terenzi MG, Marino-Neto J, et al. Feeding behavior after injection of alpha-adrenergic receptor agonists into the median raphe nucleus of food-deprived rats. Physiol Behav 2012;105(2):220–9. Sotelo C, Cholley B, Mestikawy S, Gozlan H, Hamon M. Direct immunohistochemical evidence of the existence of 5-HT1A autoreceptors on serotoninergic neurons in the midbrain raphe nuclei. Eur J Neurosci 1990;2:1144. Takase LF, Nogueira MI. Patterns of fos activation in rat raphe nuclei during feeding behavior. Brain Res 2008;1200:10–8. Vertes RP, Fortin WJ, Crane AM. Projections of median raphe nucleus of the rat. J Comp Neurol 1999;407:555–82. Walther DJ, Bader M. A unique central tryptophan hydroxylase isoform. Biochem Pharmacol 2003;66:1673–80. Wirtshafter D. The control of ingestive behavior by the median raphe nucleus. Appetite 2001;36:99–105. Wirtshafter D, Krebs JC. Control of food intake by kainate/quisqualate receptors in the median raphe nucleus. Psychopharmacology (Berl) 1990;101:137–41. Wirtshafter D, Trifunovic R. Stimulation of ingestive behaviors following injections of excitatory amino acid antagonists into the median raphe nucleus. Pharmacol Biochem Behav 1988;30:529–33. Wirtshafter D, Stratford TR, Pitzer MR. Studies on the behavioral activation produced by stimulation of GABAB receptors in the median raphe nucleus. Behav Brain Res 1993;59(1–2):83–93.
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Hall H, Lundkvist C, Halldin C, Farde L, Pike VW, Mccarron JA, et al. Autoradiographic localization of 5HT1A receptors in the post-mortem human brain using [3H] WAY-100635 and [11C] WAY 100635. Brain Res 1997;745:96–108. Higgins GA, Elliot PJ. Differential behavioural activation following intra-raphe infusion of 5HT1A receptor agonists. Eur J Pharm 1991;193:351–6. Hopwood SE, Stamford JA. Noradrenergic modulation of serotonin release in rat dorsal and median raphe nuclei via alpha 1 and alpha 2A adrenoceptors. Neuropharmacology 2001;41:433–42. Hornung JP. The human raphe nuclei and the serotonergic system. J Chem Neuroanat 2003;26:331–43. Judge SJ, Gartside SE. Firing of 5-HT neurones in the dorsal and median raphe nucleus in vitro shows differential alpha1-adrenoceptor and 5-HT1A receptor modulation. Neurochem Int 2006;48:100–7. Kia HK, Miquel MC, Brisorgueil MJ, Daval G, Riad M, El Mestikawy S, et al. Immunocytochemical localization of serotonin(1A) receptors in the rat central nervous system. J Comp Neurol 1996;365:289–305. Klitenick MA, Wirtshafter D. Comparative studies of the ingestive behaviors produced by microinjections of muscimol into the midbrain raphe nuclei of the ventral tegmental area of the rat. Life Sci 1988;42:775–82. Klitenick MA, Wirtshafter D. Elicitation of feeding, drinking and gnawing following microinjections of muscimol into the median raphe nucleus of rats. Behav Neural Biol 1989;51:436–41. Lechin F, DIJS BVD, Hernandez-Adrian G. Dorsal raphe vs. median raphe serotonergic antagonism. Anatomical, physiological, behavioral, neuroendocrinological, neuropharmacological and clinical evidences: relevance for neuropharmacological therapy. Prog Neuropsychopharmacol Biol Psychiatry 2006;30:565–85. Lucki I. The spectrum of behaviors influenced by serotonin. Biol Psychiatry 1998;44:151–62. Maidel S, Lucinda AM, Aquino VW, Faria MS, Paschoalini MA. The adrenaline microinjection into the median raphe nucleus induced hypophagic effect in rats submitted to food restriction regimen. Neurosci Lett 2007;422:123–7. Mansur SS, Terenzi MG, Marino-Neto J, Marino-Neto J, Paschoalini MA. Changes in food intake and anxiety-like behaviors after clonidine injected into the median raphe nucleus. Behav Brain Res 2010;212:71–7.
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Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
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Eduardo Simão da Silva ⁎, Rafael Appel Flores, Elisa Carolina Cella, Brunno Rocha Levone, Ana Paula Taschetto, Larissa Kochenborger, Mariana Graciela Terenzi, Moacir Serralvo Faria, Marta Aparecida Paschoalini
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Article history: Received 29 March 2014 Received in revised form 11 June 2014 Accepted 15 June 2014 Available online xxxx
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Keywords: Median raphe nucleus α1-Adrenoceptor Food intake Rat
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Previous studies have shown that the blockade of α1-adrenoceptors in the median raphe nucleus (MnR) of free-feeding animals increases food intake. Since there is evidence for the presence of α1A-, α1B- and α1Dadrenoceptors in the MnR of rats, this study investigated the involvement of MnR α1-adrenoceptor subtypes in the control of feeding behavior, looking for possible differences on the role of each α1-adrenoceptor in feeding. Male adult rats weighing 280–300 g with guide cannulae chronically implanted above the MnR were injected with antagonists of α1A- (RS100329, 0, 2, 4 or 20 nmol), α1B- (Rec 15/2615, 0, 2, 4 or 20 nmol) or α1Dadrenoceptor (BMY 7378, 0, 2, 4 or 20 nmol). Subsequently, behavioral evaluation of ingestive and noningestive parameters was monitored for 1 h and the amount of food and water ingested was assessed for 4 h. The highest dose (20 nmol) of RS100329 and BMY 7378 increased food intake, feeding duration and frequency, and decreased the latency to start feeding. During the second hour 2 nmol dose of Rec 15/2615 increased food intake and all doses of BMY 7378 decreased water intake. No behavioral alterations were observed during the fourth hour. The results corroborate previous work from our lab in which we describe the involvement of α1-adrenoceptors of MnR on food intake control. Moreover, we show evidence that α1A- and α1D-adrenoceptors mediate feeding responses to adrenaline injections and that the behavioral modifications are of considerable duration, persisting up to 2 h after injection of the antagonists. © 2014 Published by Elsevier Inc.
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1. Introduction
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The raphe nuclei are distributed rostro-caudally throughout the brainstem and present distinct neurochemical, morphological and projection characteristics (Wirtshafter, 2001; Adell et al., 2002; Hornung, 2003; Walther and Bader, 2003; Mokler et al., 2009; Takase and Nogueira, 2008). The median raphe nucleus (MnR) and the dorsal raphe nucleus send numerous serotonergic fibers to prosencephalic structures (Lucki, 1998; Hornung, 2003; Mokler et al., 2009). While the dorsal raphe nucleus innervates mainly the amygdala, ventral hippocampus and striatum (Azmitia and Segal, 1978), the MnR innervates preferentially the dorsal hippocampus, medial septum, nucleus accumbens, ventral tegmental area and several hypothalamic nuclei (Vertes et al., 1999; Lechin et al., 2006). The MnR has a diversity of neurotransmitter systems involved in the control of food intake, it is rich in 5-HT1A receptors (Sotelo et al., 1990; Kia et al., 1996; Cryan et al., 2002; Adell et al., 2002; Judge and Gartside, 2006) and the major part of these receptors is found in
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⁎ Corresponding author. Tel.: +55 48 37219352; fax: +55 48 37219672. E-mail address: [email protected] (E.S. da Silva).
serotonergic neurons acting as autoreceptors (Hall et al., 1997; Adell et al., 2002). Injection of 8-OH-DPAT (a 5-HT1A antagonist) into the MnR inhibits serotonergic neuronal firing, 5HT release at prosencephalic structures (Bonvento et al., 1992; Andrews et al., 1994; Avanzi and Brandão, 2001; Adell et al., 2002; Funk et al., 2005) and increases food intake (Currie et al., 1994). Along with 5HT receptors, GABA and glutamatergic receptors localized in the MnR also appear to participate in food intake regulation. Intra-MnR injection of kainic or quisqualic acid suppresses food intake in food-deprived rats, on the other hand antagonists of kainate/quisqualate receptors increase food intake in free-feeding rats (Wirtshafter and Krebs, 1990). Intra-MnR injection of Baclofen, a GABAB agonist, increases food and water intake and induces locomotor activity in non-deprived (free-feeding) rats (Wirtshafter et al., 1993). Data from our lab have shown that adrenaline (AD) injected into the MnR increases food intake in free-feeding rats (Maidel et al., 2007), but reduces food intake in food-deprived rats (Dos Santos et al., 2009). These opposite effects of AD were attributed to differential activation of pre-synaptic and post-synaptic α-adrenoceptors that is dependent on the nutritional state of the animal. Later data showed that MnR injections of prazosin (Mansur et al., 2011), a α1-adrenoceptor antagonist, and clonidine, a α2-adrenoceptor agonist, increased food intake in
http://dx.doi.org/10.1016/j.pbb.2014.06.010 0091-3057/© 2014 Published by Elsevier Inc.
Please cite this article as: da Silva ES, et al, Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.010
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2.1. Animals
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Male Wistar rats (weighing 270–300 g at the time of surgery) were group-housed at 22–24 °C, 12:12 light–dark cycle (lights on at 6:00 AM) with standard rodent chow and water available ad libitum. The animals were housed in groups of five per cage until the day of the experiments. After surgery, rats were housed in individual cages. The experimental procedures were conducted in compliance with the recommendations of the Ethics Committee for the use of Experimental Animals (CEUA) of the Federal University of Santa Catarina, SC, Brazil (CEUA protocol: #PP0075-2010). All efforts were made to minimize the number of animals used and their suffering.
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The rats were anesthetized (ip) with a ketamine hydrochloride (87 mg kg−1) and xylazine (13 mg kg−1) mixture and stereotaxically implanted with a unilateral stainless steel guide cannula (30G, 18 mm length). The target for this cannula was 2 mm above the MnR based on the atlas of The Rat Brain (Paxinos and Watson, 2007). The following coordinates from bregma were used: AP = −7.8 mm; L = 3.0 mm; and DV = 7.0 mm from the surface of the skull, at an angle of 20° from the vertical plane to avoid the sagittal sinus and the cerebral aqueduct. The cannula was anchored to the skull with dental cement and the whole implant stabilized with jeweler screws and more dental cement. A removable stylet was introduced to keep the cannula free from blockages until the day of the experiment.
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2.4.1. Behavioral assessment After they have been injected, the animals were placed for 60 min in a cage containing a known weight of standard rodent chow and a known volume of faucet water. At the end of the session, the measured difference between food and water at the beginning and at the end was taken as the amount of food or water consumed. The experiment was recorded with a webcam for subsequent behavioral analysis with the Etholog 2.25 (Ottoni, 2000). During the session the duration (time spent in a given behavior during experimental time), frequency (number of episodes of a given behavior) and latencies (time in seconds to initiate a given behavior) for eating, drinking, locomotion, sniffing, immobility, rearing and grooming were evaluated. See Ribas et al. (2012) for details about these procedures and description of behaviors. In addition, food and water intake recordings were also taken 2 and 4 h post-injection.
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At the end of the experiments, the animals were deeply anesthetized and transcardially perfused with saline (0.9%) and formalin (10%). The brains were removed, kept in formalin and sliced in the coronal plane (50 μm). Sections were mounted on gelatinized slides and stained with cresyl violet. The cannula placements were identified under a microscope by comparison of the sections with photographs and diagrams of The Rat Brain atlas (Paxinos and Watson, 2007). Only data from rats with cannula correctly placed in the MnR were included in the study (approximately 80% of the total of implanted animals).
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Behavioral data were analyzed by one-way ANOVA followed by Duncan's post hoc. The amount of food and water consumed during the experiment was analyzed by repeated measures ANOVA. Results are expressed as mean ± standard error of the mean (SEM). When appropriate, the ANOVA was followed by Duncan's post hoc test for multiple comparisons. Only probability values of less than 5% were considered significant.
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2.3. Drugs and injections
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Injections were made using a needle (33G, 20 mm length) extending 2 mm beyond the ventral tip of the guide cannula and connected by polyethylene tubing (PE10) to a Hamilton microsyringe (1 μl) attached to an injection pump. The injected volumes (0.2 μl) were administered over a period of 60 s and a further 60 s was allowed for the solution to diffuse from the needle. RS100329 a α1A-adrenoceptor antagonist, Rec15/2615 a α1B-adrenoceptor antagonist, and BMY7378 a α1Dadrenoceptor antagonist were given at doses of 2, 4 and 20 nmol respectively, purchased from TOCRIS Biosciences (Ellisville, MO, USA) and dissolved in 0.9% sterile saline with 5% DMSO. Each animal received only one injection: a dose of one drug or the corresponding vehicle.
All animals included in the statistical analysis had their injection site verified by histological assessment. The ANOVA of repeated measures revealed that a 20 nmol dose of RS100329 (Fig. 1) significantly increased food intake in the first hour (F(6, 68) = 3.52; p = 0.03). This treatment increased both duration (F(3, 34) = 6.5429; p = 0.001) and frequency of feeding (F(3, 34) = 7.49; p = 0.0005); in addition, a decreased latency (F(3, 34) = 4.1198; p = 0.01) to start eating was observed (Table 1). The 20 nmol dose of RS100329 did not modify drinking or non-ingestive behaviors. Both lower doses of RS100329 produced no effect on any behavior registered in this study (Table 2). Water intake was not significantly altered at any interval studied.
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Animals were acclimatized to the feeding recording chamber for 1 h for two consecutive days immediately before the experimental session. The experiment was designed to evaluate the effects of RS100329 (n = 10 for each dose), Rec 15/2615 (n = 10 for each dose) or BMY7378 (n = 9 for each dose) injection (0, 2, 4 or 20 nmol) into the MnR on ingestive and non-ingestive behaviors during the first hour. The ingestive behavior was also evaluated 2 and 4 h after drug injection.
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free-feeding rats (Mansur et al., 2010), supporting the hypothesis of a differential activation by AD. On the other hand, MnR injections of phenylephrine, a α1-adrenoceptor agonist, decreased food intake in food-deprived rats while clonidine had no effect (Ribas et al., 2012). Collectively, these results allowed us to conclude that in free-feeding animals, the activation of MnR α1-adrenoceptor receptors stimulates an inhibitory pathway on food intake. The activity of this inhibitory pathway appears to be reduced in food-deprived conditions. MnR neurons receive afferents from the locus coeruleus, lateral tegmental area, A1/A2 cell group and AD C1/C2 medullary nuclei (Hopwood and Stamford, 2001; Cryan et al., 2002; Adell et al., 2002; Lechin et al., 2006) and express mRNA of α1-adrenoceptor subtypes α1A, α1B and α1D (Day et al., 1997). There is evidence that a noradrenergic input to the MnR exerts tonic facilitatory control of 5-HT release through α1-adrenoceptors (Adell and Artigas, 1999). Given the presence of α1-adrenoceptor subtypes in the MnR, the aims of our work are to study the effect of various α1-adrenoceptor antagonists injected into the MnR on feeding behavior and to determine the duration of such effects by prolonging the period of observation of food and water intake used in previous studies.
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Fig. 1. Food intake pattern after MnR injection of vehicle (white bars), 2 (light gray bars), 4 (dark gray bars) or 20 (black bars) nmol of RS100329, Rec15/2615 or BMY 7871 in free-feeding rats. Values are mean ± SEM. *p ≤ 0.05 compared to vehicle treatment (repeated measures ANOVA followed by Duncan's post hoc test).
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The 20 nmol dose of BMY7378 (Fig. 1) significantly increased food in202 take in the first hour (F(6, 56) = 6.17; p = 0.003) and evoked significant 203 changes in this behavior increasing both duration (F(3, 29) = 9.23; 204 p = 0.0001) and frequency (F(3, 29) = 3.6, p = 0.02) and decreasing 205 latency (F(3, 29) = 6.9, p = 0.001) to start eating (Table 1). Non206 ingestive behaviors and drinking were not altered during the first hour. 207 All doses of α1D-adrenoceptor antagonist significantly decreased 208 water intake on the second hour after injection (Fig. 2; F(6, 56) = 209 Q10 9.17; p = 0.0003). No significant alterations in food or water intake 210 were observed on the fourth hour. BMY7378 in 2 or 4 nmol dose pro211 duced no effect on other behaviors registered in this study. 4. Discussion
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The present study shows that blockade of α1A- and α1Dadrenoceptors increases food intake approximately 28 min after drug injection. This response is not due to increased inespecific motor activity since the duration and frequency of non-ingestive behaviors remained unchanged. The behavioral response was characterized by increased duration and frequency and decreased latency to start feeding. Previous work showed that an α1-adrenoceptor antagonist, prazosin, injected in the MnR produced similar results (Mansur et al., 2011). Our work suggests that the prazosin effect observed in free-feeding rats was produced by blockade of α1A- and α1D-adrenoceptors. Conversely, the activation of α1-adrenoceptor in food-deprived rats resulted in a decrease in the feeding response after re-feeding (Ribas et al., 2012). Furthermore, the activation of α2-adrenoceptors results in hyperphagia in free-feeding animals (Mansur et al., 2010). This result could be attributed to an inhibition of AD release from presynaptic stores, thus removing the α1-adrenergic stimulatory tone on serotonergic neurons within the
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Interestingly, in the first hour of the experimental recording, the injection of a 4 nmol dose of Rec 15/2615, an α1B-adrenoceptor antagonist, significantly increased feeding duration (F(3, 36) = 5. 05; p = 0.005) (Table 1). In the same time interval, food intake remained unchanged as well as the frequency and the latency to start eating. During the second hour after drug injection, the 2 nmol dose of Rec 15/2615 significantly increased food intake alone (Fig. 1, F(6, 80) = 10.2, p = 0,0001), all other parameters of feeding behavior remained unchanged. No significant difference in feeding behavior was observed on the fourth hour of experimental session at any dose. Drinking and non-ingestive behaviors were not affected by drug treatments during the experimental period.
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MnR, since α2-adrenoceptor seems to exert a tonic inhibitory influence on 5-HT release in the MnR (Adell and Artigas, 1999). Taken together these data suggest that the adrenergic receptors within MnR stimulate a tonically inhibitory influence that restrains food intake under freefeeding conditions; the intensity of this inhibitory mechanism seems to decline as food availability decreases. One important issue to address related to feeding effects mediated by α1D-adrenoceptors, is that BMY 7378 is also a 5HT1A partial agonist. Thus, the increase in feeding observed in our experiments could be due to BMY 7378 acting through serotonergic receptors since it has been reported that 5HT1A activation within the MnR resulted in hyperphagia (Currie and Coscina, 1993). In fact, Higgins and Elliot (1991) showed that 8OH-DPAT, a 5HT1A agonist, as well as BMY 7378 injections into the MnR increased locomotion. However, the dose of BMY 7378 used to produce effects similar to those caused by 8-OH-DPAT was higher than the one used in our study. Thus, the dose herein used may not be high enough to activate 5HT1A receptors therefore leading to the suggestion that the feeding effects are due to activation of α1D-adrenoceptors. The extended period of observation of 2 and 4 h in our experiments revealed that α1B-adrenoceptors also have a delayed role in the control of food intake. In addition to the first hour of hyperphagia observed with α1A- and α1D-adrenoceptor antagonist administration, we also identified a late increase in food intake induced by α1B-adrenoceptor blockade. Although delayed and observed only with the lowest dose, the relevance of this mechanism could be indicated by the magnitude of the feeding response, similar to that evoked by prazosin or activation of α1A- and α1D-adrenoceptor subtypes. If this experimental design had been used in previous work, the feeding response evoked by α1Badrenoceptor might also have been seen after prazosin treatment. A possible explanation for the delayed feeding response of α1Badrenoceptor blockade is that, unlike α1A- and α1D-adrenoceptors, the signaling pathway that is being inhibited by Rec 15/2615 is a protein Gq coupled to phospholipase C. While the α1A- and α1D-adrenoceptors directly open Ca2 + channels in the membrane, α1B-adrenoceptors promote the formation of diacylglycerol and phosphatidylinositol 3-phosphate that, in turn, opens Ca2 + channels. Thus, blockade of α1B-adrenoceptors interrupts the trigger of an ongoing cascade that takes time to fully develop. However, further investigation is necessary to verify that assertion. Rec 15/2615 increased the duration of feeding for 1 h, without food intake (Table 1). Feeding frequency and the latency to start feeding were not affected by the blockade of α1B-adrenoceptors. The increase in feeding duration may be related to an intensification of oral exploration, or, on the other hand, oral motor activity could be affected by the treatment. In this situation, the animals could spend more time with food in the mouth before swallowing it, thus the prolonged duration of each eating episode significantly increased feeding duration. Previous studies have shown that rats spent more time chewing wood blocks
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26 232 0.6 28 381 0.6
0 nmol
2 nmol
0 3600 0 3.1 ± 3.1 3264 ± 335 0.1 ± 0.1
26 2471 1.1 1.5 3273 0.1
± ± ± ± ± ±
4 nmol 11 435 0.3 1.5 326 0.1
226 1794 3.2 38 2150 1.5
± ± ± ± ± ±
20 nmol 77 429 1.3 19 556 0.6
353 1723 2.7 0.3 3315 0.1
± ± ± ± ± ±
84⁎ 340⁎ 0.8⁎ 0.3 284 0.1
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Table 2 Effects of intra-MnR injection of RS100329, Rec15/2615 or BMY 7871 on non-ingestive behaviors in free-feeding rats. RS100329
t2:4
t2:20 t2:21
N
0 nmol
Rec 15/2615
Values are mean ± SEM. Duration and latency are expressed in seconds and frequency is expressed as number of events. ⁎ p ≤ 0.05 compared to vehicle treatment (one-way ANOVA followed by Duncan's post hoc test).
t2:3
t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19
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Table 1 Effects of intra-MnR injection of RS100329, Rec15/2615 or BMY 7871 on ingestive behaviors in free-feeding rats.
0 nmol Sniffing
Immobility
Rearing
Grooming
Locomotion
Duration Latency Frequency Duration Latency Frequency Duration Latency Frequency Duration Latency Frequency Duration Latency Frequency
270 4 38 2888 78 22 45 29 12 325 20 24 55 4 25
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Rec 15/2615 2 nmol
35 1 3 59 25 3 6 10 1 23 6 2 5 2 2
485 5 54 2538 107 26 81 342 14 340 691 14 91 0 33
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4 nmol 92 1 10 182 38 3 28 269 4 70 450 4 15 0 6
464 2 54 2542 242 32 80 100 21 421 422 20 75 1 27
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
20 nmol 73 1 4 135 123 5 15 83 3 86 272 4 11 0 3
507 2 57 2032 84 27 73 288 24 479 93 17 100 1 39
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0 nmol 87⁎ 1 12 278 40 2 49 197 8 79 60 2 30 0 12
345 4 47 2736 88 27 55 18 13 339 24 24 69 4.1 31
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2 nmol 82 1 10 139 26 5 12 3 3 29 7 2 14 1.9 5
Values are mean ± SEM. Duration and latency are expressed in seconds and frequency is expressed as number of events. ⁎ p ≤ 0.05 compared to vehicle treatment (one-way ANOVA followed by Duncan's post hoc test).
716 2 65 2241 121 27 59 21 17 437 56 29 127 0.7 50
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
P
20 nmol
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
552 1.8 64 2207 125 24 111 8 24 510 14 33 110 0.7 43
4 nmol 84 0.3 13 160 60 4 10 6 2 81 13 5 30 0.5 9
508 2 55 2095 441 25 69 31 28 425 51 33 110 0.5 40
138 0.4 9 128 242 5 13 10 6 76 12 7 21 0.3 6
BMY 7871
R O ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
89 0.4 11 147 24 3 28 3 5 98 8 8 20 0.3 7
0 nmol 537 2.7 112 2381 57 37 228 147 52 320 67 15 128 0.1 78
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
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2 nmol 97 0.6 23 219 21 4 58 135 15 62 11 3 34 0.1 18
F
513 1.9 92 2344 135 29 256 78 45 358 121 11 112 0.4 60
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4 nmol 82 0.3 18 243 31 6 85 68 13 83 61 2 20 0.4 10
628 2.6 91 2071 90 29 162 24 31 379 78 15 119 1.3 63
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
20 nmol 107 0.8 13 167 28 2 54 7 7 67 24 2 24 1.3 12
736 1.6 108 1909 127 33 201 29 43 369 86 15 175 0.9 73
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
104 0.4 13 174 34 3 40 18 8 70 27 3 28 0.9 10
E.S. da Silva et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
Please cite this article as: da Silva ES, et al, Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.010
t1:1 t1:2
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Fig. 2. Water intake pattern after MnR injection of vehicle (white bars), 2 (light gray bars), 4 (dark gray bars) or 20 (black bars) nmol of RS100329, Rec15/2615 or BMY 7871 in free-feeding rats. Values are mean ± SEM. *p ≤ 0.05 compared to vehicle treatment (repeated measures ANOVA followed by Duncan's post hoc test).
after injection of 8-OH DPAT (Dourish et al., 1985) and muscimol (Klitenick and Wirtshafter, 1989) into the MnR. Klitenick and Wirtshafter (1989) attribute these results to an “energizing” effect of 280 oral behaviors. The authors claim that this result appears to be consis281 tent with the idea that MnR is an integral part of a system of non282 specific behavioral activation. We suggest that adrenergic receptors 283 could also be part of this circuit, however we did not observe statistical284 ly significant changes in the duration or frequency of behaviors that in285 dicate the intensity of motor activity (vertical exploration, locomotion, 286 grooming). Thus, we conclude that the increase in eating behavior 287 may be due to specific intensified oral behavior responses. 288 Water intake was significantly decreased 2 h after α1D-adrenoceptor 289 blockade. In contrast, previous results from our lab showed that MnR 290 injections of 20 nmol AD decreased the latency to start drinking (dos 291 Santos et al., 2009). Taken together these suggest that the circuit in 292 which AD receptors take part could also control water intake. However, 293 phenylephrine or clonidine evoked no change in water intake in 294 food-deprived rats at least for 30 min after drug injections (Ribas et al., 295 2012). Several studies have shown that serotonergic and non296 serotonergic systems participate in the control of food and water intake 297 (Wirtshafter, 2001). The participation of GABAA and GABAB receptors 298 within the MnR were revealed by increases seen in food and water intake 299 induced by the injection of GABA agonists, muscimol and baclofen 300 (Klitenick and Wirtshafter, 1988; Wirtshafter et al., 1993). In addition, 301 intra-MnR treatment with glutamatergic antagonists increased feeding 302 and drinking (Wirtshafter and Krebs, 1990; Wirtshafter and Trifunovic, 303 1988). Meanwhile, 5HT1A activation by 8OH-DPAT injected into the 304 MnR appears to cause alteration in food rather than water intake 305 (Currie and Coscina, 1993; Currie et al., 1994; Fletcher, 1991; Fletcher 306 and Coscina, 1993). In that way, the AD circuit within the MnR may be 307 interacting with 5HT, GABA or glutamate neurons to control mainly 308 food intake, while the participation of 5HT neurons as mediators of the 309 AD effects on water intake could be considered minor. 310 The effects of α-adrenoceptor antagonists on ingestive behavior 311 observed in our study are confined to the MnR since previous work 312 showed that the effects of non-specific agonist and antagonists are in313 duced only with injections placed within the MnR but not in the vicinity 314 (Ribas et al., 2012; Mansur et al., 2011). 315 Intriguingly, instead of a feeding response less intense than that 316 evoked by prazosin, a full antagonist of α-adrenoceptors, the blockade 317 of individual α-adrenoceptor subtypes resulted in a feeding response 318 increase that was similar to that induced by prazosin. This may be ex319 plained by the fact that all three receptor subtypes' signaling converges 2+ 320 Q12 onto similar modifications of Ca concentrations. 321 The present work shows the involvement of α1A- and α1D322 adrenoceptors present in the MnR in the rapid control of food intake. 323 It also demonstrates a delayed control on food intake by the blockade of 324 α1B-adrenoceptor that resulted in hyperphagia. We further demonstrate
Acknowledgments
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We would like to thank the technical staff from LAMEB2, Mrs. Chirle Ferreira, Mr. Demétrio Gomes Alves and Ms. Emily Daiana dos Santos for their technical assistance. This study was supported by CNPq research grant to M. A. Paschoalini and research fellowship to E. S. Silva.
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Please cite this article as: da Silva ES, et al, Blockade of median raphe nucleus α1-adrenoceptor subtypes increases food intake in rats, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.010
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