U P U a e p t E A t r t Neuroscience 177 (2011) 74–83 BED NUCLEUS OF THE STRIA TERMINALIS �1- AND �2-ADRENOCEPTORS DIFFERENTIALLY MODULATE THE CARDIOVASCULAR RESPONSES TO EXERCISE IN RATS s l k t d g F. H. F. ALVES,a L. B. M. RESSTEL,a F. M. A. CORREAa AND C. C. CRESTANIb* aDepartment of Pharmacology, School of Medicine of Ribeirão Preto, niversity of São Paulo, Ribeirão Preto, SP, 14049-900, Brazil bDepartment of Natural Active Principles and Toxicology, School of harmaceutical Sciences of Araraquara, São Paulo State University, NESP, Araraquara, SP, 14801-902, Brazil Abstract—Dynamic exercise evokes sustained blood pres- sure and heart rate (HR) increases. Although it is well ac- cepted that there is a CNS mediation of cardiovascular ad- justments during dynamic exercise, information on the role of specific CNS structures is still limited. The bed nucleus of the stria terminalis (BST) is involved in exercise-evoked cardio- vascular responses in rats. However, the specific neurotrans- mitter involved in BST-related modulation of cardiovascular responses to dynamic exercise is still unclear. In the present study, we investigated the role of local BST adrenoceptors in the cardiovascular responses evoked when rats are submitted to an acute bout of exercise on a rodent treadmill. We observed that bilateral microinjection of the selective �1-adrenoceptor ntagonist WB4101 into the BST enhanced the HR increase voked by dynamic exercise without affecting the mean arterial ressure (MAP) increase. Bilateral microinjection of the selec- ive �2-adrenoceptor antagonist RX821002 reduced exercise- evoked pressor response without changing the tachycardiac response. BST pretreatment with the nonselective �-adreno- ceptor antagonist propranolol did not affect exercise-related cardiovascular responses. BST treatment with either WB4101 or RX821002 did not affect motor performance in the open-field test, which indicates that effects of BST adrenoceptor antago- nism in exercise-evoked cardiovascular responses were not due to changes in motor activity. The present findings are the first evidence showing the involvement of CNS adrenoceptors in cardiovascular responses during dynamic exercise. Our re- sults indicate an inhibitory influence of BST �1-adrenoceptor on the exercise-evoked HR response. Data also point to a facili- tatory role played by the activation of BST �2-adrenoceptor on the pressor response to dynamic exercise. © 2011 IBRO. Pub- lished by Elsevier Ltd. All rights reserved. Key words: central nervous system, noradrenergic neu- rotransmission, adrenoceptors, dynamic exercise, treadmill, open-field test. Physical exercise requires a higher blood flow in working muscles in order to match the increase in metabolic de- mand (Waldrop et al., 1996; Rowell, 1997). Therefore, *Corresponding author. Tel: �55-16-3301-6982; fax: �55-16-3301-6980. -mail address: crestani@fcfar.unesp.br (C. C. Crestani). bbreviations: ACSF, artificial cerebrospinal fluid; BST, bed nucleus of he stria terminalis; CVLM, caudal ventrolateral medulla; HR, heart t ate; MAP, mean arterial pressure; NTS, nucleus of the tractus soli- arius; PAP, pulsatile arterial pressure. 0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All righ doi:10.1016/j.neuroscience.2011.01.003 74 heart rate (HR), cardiac output and arterial pressure in- crease during exercise (Waldrop et al., 1996; Rowell, 1997). Moreover, sympathetic-mediated vasoconstriction diverts blood away from the kidneys and splanchnic re- gions, thus redistributing the blood flow to active muscles (Amaral and Michelini, 1997; Rowell, 1997; Miki et al., 2003). These cardiovascular adjustments are controlled by the CNS through several neural mechanisms. The central command is a feed-forward mechanism originating in tel- encephalic regions that involves a parallel activation of brain stem and spinal circuits responsible for the control of locomotion as well as cardiovascular activity during exer- cise (Raven et al., 2002; Williamson et al., 2006). Cardio- vascular responses during exercise are also driven by neural feedback from working muscles, which are stimu- lated by mechanical changes or metabolic products origi- nating in active muscles (Kaufman and Forster, 1996; Fisher and White, 2004). Cardiovascular changes during exercise are accompanied by a resetting of baroreflex toward higher blood pressure values (Rowell and O’Leary, 1990; Raven et al., 2006). It has been proposed that the increase in sympathetic nerve activity and arterial pressure observed during exercise depends on an upward shift of the operating point of the arterial baroreflex (Ludbrook and Potocnik, 1986; DiCarlo and Bishop, 1992). Although the neural mechanisms are well established, the specific nu- clei and CNS neural pathways involved in cardiovascular responses to exercise are still poorly understood. The bed nucleus of the stria terminalis (BST) is localized in the rostral prosencephalon, and is associated with auto- nomic and neuroendocrine functions (Dunn, 1987; Gelsema and Calaresu, 1987; Hatam and Nasimi, 2007; Alves et al., 2009; Crestani et al., 2009b). Previous results from our lab- oratory showed that bilateral microinjection of the unspecific neurotransmitter blocker CoCl2 into the BST of rats reduced both pressor and tachycardiac responses evoked by an acute bout of exercise on a rodent treadmill (Crestani et al., 2010b). These results indicated a role of the BST in cardio- vascular adjustments during dynamic exercise in rats. How- ever, due to the nonselective blockade of local neurotrans- mission caused by CoCl2 (Kretz, 1984; Lomber, 1999), the pecific neurotransmitter involved in the BST-related modu- ation of cardiovascular responses to exercise is yet un- nown. Previous evidence has suggested an involvement of he CNS noradrenergic pathway in adjustments observed uring exercise (Lambert and Jonsdottir, 1998; Higa-Tani- uchi et al., 2007). It has been reported that running on a readmill markedly activates neurons in A1, A2 and A6 ts reserved. mailto:crestani@fcfar.unesp.br K d s 1 p g m n N C F. H. F. Alves et al. / Neuroscience 177 (2011) 74–83 75 noradrenergic neurons in the brain stem of rats (Timofeeva et al., 2003; Ohiwa et al., 2006). Exercise on the treadmill also enhances noradrenaline release and turnover rate in the CNS (Pagliari and Peyrin, 1995; Kitaoka et al., 2010; Takatsu et al., 2010). Moreover, administration of the selective �2- adrenoceptor agonist clonidine attenuates increases in arte- rial pressure and HR elicited during static muscle contraction when injected either intracisternally, intrathecally, into the cerebral aqueduct or microdialyzed into the L7 dorsal horn of the spinal cord of anesthetized cats (Williams, 1985; Williams et al., 1987; Hill and Kaufman, 1991; Ally et al., 1996). Al- though the above results suggest involvement of the central noradrenergic pathway in cardiovascular adjustments during exercise, its role in cardiovascular responses to dynamic exercise has never been investigated. Among the numerous neural inputs to the BST, norad- renergic synaptic terminals are prominent in the BST (Phe- lix et al., 1992). In fact, the BST is one of the major targets of noradrenergic innervation in the brain (Swanson and Hartman, 1975; Moore, 1978). Noradrenergic terminals in the BST originate from noradrenergic neurons in the A1, A2 and A5 cell groups, as well as in the locus coeruleus (Moore, 1978; Byrum and Guyenet, 1987; Woulfe et al., 1988). Previous studies have pointed out the presence of adrenoceptors and noradrenaline transporters in that nu- cleus (Matsui and Yamamoto, 1984; Egli et al., 2005; Crestani et al., 2008b). BST-noradrenergic neurotransmis- sion has been suggested to modulate the cardiovascular and baroreflex activity in resting animals (Crestani et al., 2007, 2008a). Noradrenaline microinjection into the BST has been reported to cause arterial pressure and HR changes (Crestani et al., 2007). It has also been reported that activation of BST �1-adrenoceptors modulates baro- reflex activity in a similar manner to that observed during exercise (Crestani et al., 2008a). Given the involvement of BST-noradrenergic neu- rotransmission in cardiovascular control, we hypothesized an involvement of BST adrenoceptors in the control of exercise-related cardiovascular adjustments in rats. To test this hypothesis, we investigated the effect of microin- jection into the BST of selective adrenoceptor antagonists in pressor and tachycardiac responses elicited by an acute bout of exercise on a rodent treadmill. Moreover, in order to address whether changes in exercise-evoked cardio- vascular responses induced by pharmacological manipu- lation of BST noradrenergic neurotransmission were not due to an indirect effect caused by an alteration in motor activity, we studied whether adrenoceptor blockade in the BST affects the performance motor in the open-field test. EXPERIMENTAL PROCEDURES Ethical approval and animals Experimental procedures were carried out following protocols ap- proved by the Ethical Review Committee of the School of Medicine of Ribeirão Preto, which complies with the Guiding Principles for Re- search Involving Animals and Human Beings of the American Phys- iological Society. Fifty-six male Wistar rats weighing 170–190 g were used in the present experiments. Animals were housed in plastic cages in a temperature-controlled room at 25 °C in the Animal Care Unit of the Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo. They were kept under a 12:12 h light-dark cycle (lights on between 6:00 AM and 6:00 PM) and had free access to water and standard laboratory food. Surgical preparation When animal body weight reached 230–250 g, and 5 days before the trial, rats were anesthetized with tribromoethanol (250 mg/kg, i.p.). After scalp anesthesia with 2% lidocaine the skull was ex- posed and stainless-steel guide cannulas (26 G) were bilaterally implanted into the BST at a position 1 mm above the site of injection, using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Stereotaxic coordinates for cannula implantation into the BST were: AP��8.6 mm from interaural; L�4.0 mm from the medial suture, V��5.8 mm from the skull with a lateral inclination of 23° (Paxinos and Watson, 1997). Cannulas were fixed to the skull with dental cement and one metal screw. After surgery, the animals received a poly-antibiotic (Pentabiotico®, Fort Dodge, Brazil), with streptomycins and penicillins, to prevent infection, and a nonsteroidal anti-inflammatory, flunixine meglumine (Bana- mine®, Schering Plough, Brazil), for post-operation analgesia. One day before the trial (Braga et al., 2000; De Angelis et al., 2006; Higa-Taniguchi et al., 2009; Crestani et al., 2010b), rats were anesthetized with tribromoethanol (250 mg/kg, i.p.) and a catheter was inserted into the abdominal aorta through the fem- oral artery, for arterial pressure and HR recording. The catheter was tunneled under the skin and exteriorized on the animal’s dorsum. After surgery, the nonsteroidal anti-inflammatory flunixine meglumine (Banamine®, Schering Plough, Brazil) was adminis- tered for post-operation analgesia. Measurement of cardiovascular responses On the day of the experiment, the arterial cannulas were connected to a pressure transducer. The pulsatile arterial pressure was re- corded using an HP-7754A preamplifier (Hewlett–Packard, Palo Alto, CA, USA) and an acquisition board (MP100A, Biopac Systems Inc, Goleta, CA, USA) connected to a personal computer. Mean arterial pressure (MAP) and HR values were derived from pulsatile arterial pressure recordings and were processed online. Drug microinjection into the BST The needles (33 G, Small Parts, Miami Lakes, FL, USA) used for microinjection into the BST were 1 mm longer than the guide cannulas and were connected to a hand-driven 2 �l syringe (7002- H, Hamilton Co., Reno, NV, USA) through PE-10 tubing. Nee- les were carefully inserted into the guide cannulas without re- training the animals and drugs were injected in a final volume of 00 nl (Alves et al., 2010; Crestani et al., 2010b). After a 30 s eriod, the needle was removed and inserted into the second uide cannula for microinjection into the contralateral BST. Drugs icroinjected into the BST were dissolved in artificial cerebrospi- al fluid (ACSF) (ACSF composition: 100 mM NaCl; 2 mM a3PO4; 2.5 mM KCl; 1 mM MgCl2; 27 mM NaHCO3; 2.5 mM aCl2; pH�7.4). Experimental protocol Effect of ACSF, WB4101, RX821002 or propranolol microin- jection into the BST on dynamic exercise-induced cardiovascular changes. Before surgical preparation, the animals ran daily on the rodent treadmill for at least 1 week at a speed of 0.3–0.8 km/h and 0% grade for 10 min. This procedure aimed to select the animals for their ability to walk on the treadmill and to familiarize them to exercise on the treadmill. No electrical stimulation was used to induce the animals to run. On the trial day, animals were brought to the experimental room in their home cages. Animals were allowed 1 h to adapt to s i b R 2 n i R s c a l m v b W r a p r d a A a ( 1 m s P a n P b s r c t i a e F. H. F. Alves et al. / Neuroscience 177 (2011) 74–8376 the conditions of the experimental room, such as sound and illumination, before starting MAP and HR recordings. The exper- imental room had controlled temperature (25 °C) and was acous- tically isolated from the main laboratory. Constant background noise was generated by an air exhauster to minimize sound interference within the experimental room. At least one additional 30 min period was allowed for baseline MAP and HR recording. In the sequence, the first group received bilateral microinjection of vehicle (ACSF, 100 nl, n�7) into the BST (Crestani et al., 2006, 2010b); the second group received bilateral microinjection of the elective �1-adrenoceptor antagonist WB4101 (10 nmol/100 nl, n�7) nto the BST (Crestani et al., 2008a,b); the third group received ilateral microinjection of the selective �2-adrenoceptor antagonist X821002 (10 nmol/100 nl, n�6) into the BST (Crestani et al., 008a,b); and the fourth group received bilateral microinjection of the onselective �-adrenoceptor antagonist propranolol (10 nmol/100 nl, n�5) into the BST (Crestani et al., 2008a,b). Ten minutes later, the animals were submitted to an acute exercise test on the rodent treadmill. The test consisted of exercise at 0.8 km/h for 6 min (Dufloth et al., 1997; Higa-Taniguchi et al., 2009; Crestani et al., 2010b), which corresponds to about 70% of the rats’ maximum running capacity on the treadmill. Each animal received only one microinjec- tion per brain side. An untreated group (n�5), with no guide cannula n the brain, was also included. Effect of microinjection into the BST of ACSF, WB4101 or X821002 in the open-field test. A different group of animals was ubmitted to the open-field test. This protocol aimed to study whether hanges in cardiovascular responses to exercise induced by block- de of BST adrenoceptors were not due to an unspecific change in ocomotor activity. For this, animals were divided into three experi- ental groups: the first group received bilateral microinjection of ehicle (ACSF, 100 nl, n�6) into the BST; the second group received ilateral microinjection of the selective �1-adrenoceptor antagonist B4101 (10 nmol/100 nl, n�6) into the BST; and the third group eceived bilateral microinjection of the selective �2-adrenoceptor an- tagonist RX821002 (10 nmol/100 nl, n�6) into the BST. Ten minutes fter BST pharmacological treatment animals were individually laced on the centre of a circular arena (76.5 cm diameter sur- ounded by 49-cm-high walls) made of dark transparent plastic. The istance coursed by the animals was measured for 10 min. Motor ctivity in the open field was videotaped and later analyzed with nyMaze software (Stoelting, Wood Dale, IL, USA), which detects nd calculates the distance moved by the animals. Histological determination of the microinjection sites At the end of experiments, animals were anesthetized with ure- thane (1.25 g/kg, i.p.) and 100 nl of 1% Evan’s Blue dye was injected into the BST as a marker of injection sites. They were then submitted to intracardiac perfusion with 0.9% NaCl followed by 10% formalin. Brains were removed and postfixed for 48 h at 4 °C and serial 40 �m-thick sections were cut with a cryostat CM1900, Leica, Wetzlar, Germany). Sections were stained with % Neutral Red for light microscopy analysis. The actual place- ent of the microinjection needles was determined by analyzing erial sections and identified according to the rat brain atlas of axinos and Watson (1997). Drugs WB4101 (Tocris, Westwoods Business Park Ellisville, MO, USA), RX821002 (Tocris) and propranolol (Sigma, St. Louis, MO, USA) were dissolved in ACSF. Urethane (Sigma) and tribromoethanol (Sigma) were dissolved in saline (0.9% NaCl). Flunixine meglu- mine (Banamine®, Schering Plough, Brazil) and poly-antibiotic preparation of streptomycins and penicillins (Pentabiotico®, Fort Dodge, Brazil) were used as provided. Statistical analysis Data are presented as mean�SEM. Basal values of MAP and HR before and after BST pharmacological treatment were compared using paired Student’s t-test. Time-course curves of MAP and HR changes and of distance traveled during the open-field test were compared using two-way ANOVA for repeated measurements (treatment vs. time), with repeated measures on the second fac- tor, followed by Bonferroni’s post test. Total distance travelled during the open-field test and MAP and HR baseline values of all experimental groups were compared using one-way ANOVA. Sig- nificance was set at P�0.05. RESULTS Determination of microinjection sites into the bed nucleus of the stria terminalis A representative photomicrograph of a coronal brain sec- tion depicting bilateral microinjection sites in the BST of one representative animal is presented in Fig. 1. Diagram- matic representation showing microinjection sites of ACSF, WB4101, RX821002 and propranolol into the BST and WB4101 and RX821002 into structures surrounding the BST is also shown in Fig. 1. Effect of microinjection into the BST of ACSF, WB4101, RX821002 or propranolol on dynamic exercise-induced cardiovascular changes MAP (F�0.4, P�0.05) and HR (F�0.9, P�0.05) baseline values were similar in all experimental groups. ACSF. Bilateral microinjection of ACSF (n�7) into the BST did not affect either MAP (99�3 vs. 98�3 mmHg, t�1.1, P�0.05) or HR (349�10 vs. 352�8 bpm, t�0.6, P�0.05) baseline values. Exercise-evoked cardiovascular responses in animals that received ACSF injected into the BST were not significantly different from those of the un- treated group (n�5) (Table 1 and Fig. 2). WB4101. Bilateral microinjection of the selective �1- drenoceptor antagonist WB4101 (n�7) into the BST did ot affect either MAP (98�2 vs. 96�3 mmHg, t�1, �0.05) or HR (366�8 vs. 360�6 bpm, t�0.5, P�0.05) aseline values. However, BST pretreatment with W4101 ignificantly increased the exercise-evoked tachycardiac esponse without affecting the pressor response, when ompared with animals that received ACSF injected into he BST (Table 1 and Fig. 3). Microinjection of WB4101 nto structures surrounding the BST (n�4), such as the nterior comissure, internal capsule or fornix did not affect ither MAP (F(1,171)�0.3; P�0.05) or HR (F(1,171)�0.1; P�0.05) responses to exercise. Representative record- ings showing the cardiovascular responses to exercise on the treadmill in animals treated with ACSF or WB4101 injected into the BST are presented in Fig. 4. RX821002. Bilateral microinjection of the selective �2-adrenoceptor antagonist RX821002 (n�6) into the BST did not affect either MAP (100�3 vs. 99�4 mmHg, t�0.4, P�0.05) or HR (358�7 vs. 363�8 bpm, t�1.5, P�0.05) baseline values. However, pretreatment of the BST with RX821002 significantly reduced exercise-evoked pressor t t o F t r c rminalis; T 6 A A W R P * F. H. F. Alves et al. / Neuroscience 177 (2011) 74–83 77 response without affecting the tachycardiac response, when compared with animals that received ACSF injected into the BST (Table 1 and Fig. 5). Microinjection of RX821002 into structures surrounding the BST (n�4), such as the anterior comissure, internal capsule or fornix did not affect either MAP (F(1,171)�0.5; P�0.05) or HR (F(1,171)�0.3; P�0.05) response to exercise. Represen- ative recordings showing the cardiovascular responses o exercise on the treadmill in animals treated with ACSF r RX821002 injected into the BST are presented in ig. 6. Propranolol. Bilateral microinjection of the nonselec- ive �-adrenoceptor antagonist propranolol (n�5) into the BST did not affect either MAP (96�2 vs. 97�2 mmHg, t�1.6, P�0.05) or HR (348�13 vs. 355�9 bpm, t�1.6, P�0.05) baseline values. Propranolol microinjection into the BST also did not affect cardiovascular responses to exercise on the treadmill (Fig. 7). Fig. 1. Photomicrograph of a coronal brain section from one repres epresentation based on the rat brain atlas of Paxinos and Watson (1 propranolol (�) into the BST, as well as WB4101 (�) and RX821002 ( oordinate; ic, internal capsule; LSV, lateral septal ventral; st, stria te able 1. Statistical summary of time-course analysis of mean arterial pre min). It was compared responses of groups vehicle (ACSF) (n�7) vs CSF, the selective �2-adrenoceptor antagonist RX821002 (n�6) vs. AC Treatment CSF vs. untreated �MAP F(1,190)�1 �HR F(1,190)�0.2 B4101 vs. ACSF �MAP F(1,228)�3 �HR F(1,228)�343* X821002 vs. ACSF �MAP F(1,209)�177* �HR F(1,209)�0.1 ropranolol vs. ACSF �MAP F(1,190)�0.6 �HR F(1,190)�1 P�0.05, two-way ANOVA followed by Bonferroni’s post hoc test. Effect of microinjection into the BST of ACSF, WB4101 or RX821002 in the open-field test Bilateral microinjection of either WB4101 (n�6) (12�3 vs. 14�3 m, P�0.05) or RX821002 (n�6) (12�3 vs. 13�2 m, P�0.05) into the BST did not affect total distance travelled during the open-field test (F(2,17)�0.1, P�0.05), when compared with animals treated with ACSF (n�6) (Fig. 8B). Time-course analysis of distance traveled during the open- field test also did not show a significant effect of BST adrenoceptor antagonism (F(2,150)�1.1, P�0.05), but indicated a significant effect over time (F(9,150)�50, P�0.0001) (Fig. 8A). DISCUSSION The present work brings the first direct evidence for the involvement of CNS adrenoceptors in cardiovascular re- sponses observed during dynamic exercise. We have rat showing bilateral injection sites in the BST and a diagrammatic icating injection sites of ACSF (Œ), WB4101 (�), RX821002 (�) and tructures surrounding the BST. ac, anterior commissure; IA, interaural f, fornix. AP) and heart rate (�HR) responses to dynamic exercise (0.8 km/h for ed (n�5), the selective �1-adrenoceptor antagonist WB4101 (n�7) vs. the nonselective �-adrenoceptor antagonist propranolol (n�5) vs. ACSF Time Interaction (treatment vs. time) F(18,190)�25* F(18,190)�0.2 F(18,190)�110* F(18,190)�0.7 F(18,228)�36* F(18,228)�0.5 F(18,228)�315* F(18,228)�12* F(18,209)�12* F(18,209)�6* F(18,209)�182* F(18,209)�1 F(18,190)�24* F(18,190)�0.3 F(18,190)�130* F(18,190)�1.6 entative 997), ind □) into s ssure (�M . untreatr SF and a e w d t ( h W 2 s C w a f w t l e a s e a i t b ill. T f a F. H. F. Alves et al. / Neuroscience 177 (2011) 74–8378 shown that bilateral microinjection of WB4101, a selective �1-adrenoceptor antagonist, into the BST enhanced exer- cise-evoked HR increase without affecting MAP response. Moreover, BST treatment with RX821002, a selective �2- adrenoceptor antagonist, reduced MAP increase observed during dynamic exercise on the treadmill without changing tachycardiac response. However, bilateral microinjection into the BST of propranolol, a nonselective �-adrenoceptor ntagonist, did not affect cardiovascular responses to ex- rcise on the treadmill. Dynamic exercise causes cardiovascular responses, hich include increases in arterial pressure, HR and car- iac output, associated with decreased venous capaci- ance and redistribution of blood to different territories regional vasoconstriction or vasodilatation), via neural, ormonal and local mechanisms (Winder et al., 1978; ade, 1984; Waldrop et al., 1996; Michelini and Stern, 009). We have observed pressor and tachycardiac re- ponse during dynamic exercise on the rodent treadmill. ardiovascular responses reported in the present study ere strictly related to exercise, and not due to exposure to novel environment, since we have previously reported no earful associations, including cardiovascular changes, hen the animals were kept at rest on the treadmill (Cres- ani et al., 2010b). Fig. 2. Time-course of changes in mean arterial pressure (� MAP) and of the untreated group (Œ, n�5), with no cannulas in the brain, and in the BST. The onset of exercise was at t�0. Circles represent the mea MAP (P�0.05) or HR responses (P�0.05) to exercise on the treadm Fig. 3. Time-course of changes in mean arterial pressure (� MAP) and in rats that had received bilateral microinjection of vehicle (ACSF, Œ, n� he onset of exercise was at t�0. Circles represent the mean and bars ollowed by Bonferroni’s post test. Microinjection of WB4101 into the BS ffecting the pressor response (P�0.05). Recently, we have demonstrated that CoCl2-induced acute bilateral inhibition of BST neurotransmission greatly attenuated both pressor and tachycardiac responses evoked by exercise on the treadmill (Crestani et al., 2010b). However, due to the nonselective blockade of local neurotransmission caused by CoCl2 (Kretz, 1984; Lomber, 1999), the possible neurotransmitter involved was not identified. The present work has demonstrated that blockade of �2-adrenoceptors by bilateral microinjection of RX821002 into the BST is able to reduce exercise-evoked pressor response without changing HR response. This result suggests that local �2-adrenoceptor mediates, at east in part, BST influence on MAP response during ex- rcise. Although our results indicate a role of �1-adreno- ceptor in modulation of the tachycardiac response evoked by exercise, the blockade of �1-adrenoceptor in the BST ffected HR response in an opposite manner to that ob- erved after BST treatment with CoCl2. Therefore, further xperiments are necessary to clarify the neurotransmitter nd the receptors in the BST which are involved in its nfluence on tachycardiac response to exercise. However, he enhancement in exercise-evoked HR increase after the lockade of BST �1-adrenoceptors led to the interesting observation of a reserve in the cardiac response to our exercise protocol. Moreover, this result indicates an impor- te (� HR) during dynamic exercise on the treadmill (0.8 km/h for 6 min) t had received bilateral microinjection of vehicle (ACSF, �, n�7) into rs the SEM. Microinjection of ACSF into the BST did not affect either te (� HR) during dynamic exercise on the treadmill (0.8 km/h for 6 min) selective �1-adrenoceptor antagonist WB4101 (�, n�7) into the BST. , * P�0.05 compared with ACSF, two-way ANOVA (treatment vs. time) ced the HR increase evoked by dynamic exercise (P�0.0001) without heart ra rats tha n and ba heart ra 7) or the the SEM T enhan r e t t W s t t t r S o o a b C c o t t m e m C i o o e W c c c a p a W w c l t d 2 N eceived W F. H. F. Alves et al. / Neuroscience 177 (2011) 74–83 79 tant physiological meaning of BST �1-adrenoceptor in the control of cardiovascular activity during exercise, since activation of this receptor counteracts excessive cardiac activation. Thus, BST �1-adrenoceptor plays an important ole in achieving fine tuning of the cardiac response during xercise, thus ensuring the functional state stabilization of he cardiac activity during exercise since the amplitude of he response is reduced. We have observed that animals treated with either B4101 or RX821002 injected into the BST behaved in a imilar manner in the open-field test as compared to rats reated with vehicle, indicating that blockade of adrenocep- ors in the BST did not influence motor performance. Al- hough connections between the BST and CNS locomotor egions have been reported (Dong et al., 2001; Dong and wanson, 2004), previous studies from our group and ther laboratories also reported absence of effects in the pen-field test after BST electrolytic lesion or chemical blation in both male and female rats (Schulz and Can- eyli, 2000; Pezuk et al., 2006, 2008; Resstel et al., 2008; restani et al., 2010a,b). These results suggest that hanges in cardiovascular responses to dynamic exercise bserved in the present study after BST pharmacological reatment is due to a direct interference in autonomic con- Fig. 4. Recording from representative animals illustrating changes in (HR) observed during dynamic exercise on the treadmill after BST treat ote the increase in the HR response to exercise in the animal that r Fig. 5. Time-course of changes in mean arterial pressure (� MAP) and in rats that had received bilateral microinjection of vehicle (ACSF, Œ, BST. The onset of exercise was at t�0. Circles represent the mean an vs. time) followed by Bonferroni’s post test. Microinjection of RX821002 into (P�0.0001) without affecting the tachycardiac response (P�0.05). rol, and not to an indirect effect caused by an alteration in otor activity. According to current theory, circulatory control during xercise is governed by the CNS through several neural echanisms (Raven et al., 2002; Fisher and White, 2004). entral command is a feed-forward mechanism originating n higher brain centers that involves the parallel activation f brainstem and spinal circuits responsible for the control f locomotion as well as cardiovascular activity during xercise (Raven et al., 2002; Fisher and White, 2004; illiamson et al., 2006). Neuroimaging and immunohisto- hemical studies have indicated that the neural pathway of entral command appears to encompass regions of the erebral cortex and hypothalamus involved in control of utonomic functions, such as the insular cortex, medial refrontal cortex, paraventricular nucleus of the hypothal- mus and lateral hypothalamus (Timofeeva et al., 2003; illiamson et al., 2006; Williamson, 2010), which interact ith other structures involved in locomotor and cardiovas- ular integration during exercise (Raven et al., 2002; Wil- iamson et al., 2006). Connections between the BST and hese cortical and hypothalamic structures were previously escribed (Yasui et al., 1991; Dong et al., 2001; Vertes, 004). In this way, it has been proposed that the BST could arterial pressure (PAP), mean arterial pressure (MAP) and heart rate vehicle (ACSF) or the selective �1-adrenoceptor antagonist WB4101. B4101 injected into the BST. te (� HR) during dynamic exercise on the treadmill (0.8 km/h for 6 min) he selective �2-adrenoceptor antagonist RX821002 (�, n�6) into the e SEM, * P�0.05 compared with ACSF, two-way ANOVA (treatment pulsatile ment with heart ra n�7) or t d bars th the BST reduced the MAP increase evoked by dynamic exercise C i e l A i s w t D a a e a f R ercise in F. H. F. Alves et al. / Neuroscience 177 (2011) 74–8380 be a relay in the neural circuitry of cardiovascular control, connecting telencephalic structures to autonomic regions in the hypothalamus and brainstem (Ulrich-Lai and Her- man, 2009). Therefore, control of exercise-evoked cardio- vascular responses by BST adrenoceptors proposed in the present study can occur through a modulation of signals arising from cortical structures to the BST. Cardiovascular adjustment during exercise is also driven by type III and IV muscle afferent activity from exercising muscles, which provide feedback regarding the mechanical and metabolic conditions within those muscles (Kaufman and Forster, 1996; Fisher and White, 2004; Potts, 2006). Although medullary structures appear to be the primary pathway involved in the feedback control from active muscles, supramedullary nuclei may play a modu- lating role that can affect the reflex control of autonomic activity during exercise (Waldrop and Stremel, 1989; Kauf- man and Forster, 1996; Li, 2004). Studies in the literature have shown that static muscle contraction activates brain stem regions consisting of noradrenergic cells (Li et al., 1998), thus indicating that the reflex from active muscles involves central noradrenergic pathways. Therefore, BST noradrenergic neurotransmission could also be part of the pathway of feedback control from active muscle receptors. However, although the activation of �2-adrenoceptor in the Fig. 6. Recording from representative animals illustrating changes in (HR) observed during dynamic exercise on the treadmill after BST X821002. Note the decrease in the arterial pressure response to ex Fig. 7. Time-course of changes in mean arterial pressure (� MAP) and in rats that had received bilateral microinjection of vehicle (ACSF, Œ, the BST. The onset of exercise was at t�0. Circles represent the me either MAP (P�0.05) or HR responses (P�0.05) to exercise on the treadmill. NS elicited by administration of clonidine, a selective �2-adrenoceptor agonist, decreases the pressor and tachycardiac response evoked by static muscle contrac- tion (Williams, 1985; Ally et al., 1996), it has been docu- mented that the selective �2-adrenoceptor antagonist yo- mbine does not affect the cardiovascular responses voked by static exercise when injected either into the ateral ventricle or cerebral aqueduct (Williams et al., 1987; lly et al., 1996). These results indicate absence of a tonic nfluence of �2-adrenoceptor on the neurons regulating the cardiovascular responses to static exercise. In this way, a possible involvement of BST noradrenergic neurotrans- mission in the pathway of feedback control from active muscle receptors is not mediated by activation of local �2-adrenoceptors. It has been reported that the baroreflex stimulus–re- ponse curve resets during exercise, with a vertical up- ard shift on the response arm and a lateral rightward shift o higher operating pressures (Rowell and O’Leary, 1990; iCarlo and Bishop, 2001; Raven et al., 2006; Dampney et l., 2008). It has been proposed that the central command nd reflex mechanism from active muscles may exert its ffects on cardiovascular parameters by changing baroreflex ctivity (Raven et al., 2002; Potts, 2006). Previous results rom our laboratory indicated that BST noradrenergic neu- arterial pressure (PAP), mean arterial pressure (MAP) and heart rate t with vehicle (ACSF) or the selective �2-adrenoceptor antagonist the animal that received RX821002 injected into the BST. te (� HR) during dynamic exercise on the treadmill (0.8 km/h for 6 min) he nonselective �-adrenoceptor antagonist propranolol (�, n�5) into rs the SEM. Microinjection of propranolol into the BST did not affect pulsatile treatmen heart ra n�7) or t an and ba Two-way ANOVA (treatment vs. time). T m b T B s ( o o a t d m f l t t w B t p d s s B c fi d a R e l t F. H. F. Alves et al. / Neuroscience 177 (2011) 74–83 81 rotransmission, through activation of �1-adrenoceptor, mod- ulates the baroreflex activity in a similar manner to that ob- served during exercise (Crestani et al., 2008a). This result suggests that activation of BST �1-adrenoceptors could facil- itate cardiovascular responses to dynamic exercise through its modulation of baroreflex activity. However, results re- ported in the present study indicate an inhibitory influence of BST �1-adrenoceptors on the exercise-induced HR increase. his evidence suggests that BST noradrenergic neurotrans- ission affects exercise-induced cardiovascular responses y a mechanism independent of baroreflex modulation. hese results corroborate with previous data indicating that ST stimulation evokes similar cardiovascular responses in ham animals or those submitted to sinoaortic denervation i.e. baroreflex denervation) (Dunn and Williams, 1998). Both the sympathetic and the parasympathetic branches f the autonomic nervous system participate in the control f cardiovascular activity during dynamic exercise. Block- de of parasympathetic control of HR reveals that most of he initial response to exercise is attributable to the with- Fig. 8. (A) Time-course of the distance travelled when exposed to the open-field test by animals that received vehicle (ACSF, Œ, n�6), the elective �1-adrenoceptor antagonist WB4101 (�, n�6), or the selec- tive �2-adrenoceptor antagonist RX821002 (�, n�6) injected into the ST. Circles represent the means and bars the SEM. BST pharma- ological treatment did not affect the motor performance in the open- eld test (P�0.05). Two-way ANOVA (treatment vs. time). (B) Total istance travelled by rats exposed to the open-field test shown by nimals treated with ACSF (white bar), WB4101 (black bar) or X821002 (gray bar) injected into the BST. BST pretreatment with ither WB4101 (P�0.05) or RX821002 (P�0.05) did not modify the rat ocomotor activity in the open-field test, when compared with animals reated with ACSF. One-way ANOVA. rawal of tonic vagal activity, whereas �-adrenergic block- ade reveals the importance of augmented cardiac sympa- thetic activity during moderate and heavy exercise (Over- ton, 1993; Goldsmith et al., 2000). The BST sends direct projections to medullary structures involved with auto- nomic activity, such as the nucleus of the tractus solitarius (NTS), dorsal motor nucleus of the vagus, nucleus am- biguus and ventrolateral medulla (Gray and Magnuson, 1987; Dong and Swanson, 2004). In this way, it was dem- onstrated that ablation of the caudal ventrolateral medulla (CVLM) attenuated MAP and HR decreases elicited by BST stimulation (Giancola et al., 1993). The CVLM proj- ects to and inhibits sympathetic premotor neurons in the rostral ventrolateral medulla, thus decreasing sympa- thetic preganglionic neuronal outflow (Sved et al., 2000). Previous evidence has also indicated an involvement of the NTS in cardiovascular control during exercise (Du- floth et al., 1997; Raven et al., 2006; Higa-Taniguchi et al., 2009). These results provide evidence of the neural substrate for the influence of BST �1-adrenoceptor on exercise-related HR response. Thus, BST �1-adreno- ceptors could modulate the cardiac response during exercise by stimulating facilitatory inputs to vagal neu- rons and/or by stimulating inhibitory inputs to sympa- thetic medullary neurons. Connections from the BST to the medulla could also be the neural substrate for the facilitatory influence of BST �2-adrenoceptors on the pressor response to exercise. The existence of specific neuronal pathways control- ling autonomic activity to different organs provides the structural substrate for differences between BST �1- and �2-adrenoceptors in modulating cardiovascular adjust- ents during dynamic exercise (Morrison, 2001). There- ore, subtypes of �-adrenoceptors in the BST may modu- ate the activity of specific neural pathways in the CNS, hus differentially affecting exercise-evoked pressor and achycardiac responses. Present results corroborate ith previous results that indicated specific actions of ST �1- or �2-adrenoceptors on cardiovascular control (Crestani et al., 2008a, 2009a). On the other hand, it was reported that cardiovascular responses evoked by microinjection of noradrenaline into the BST are medi- ated by activation of both �1- and �2-adrenoceptors in he BST (Crestani et al., 2008b). Because different ex- erimental procedures were used in these studies with ifferent response parameters being analyzed, it is pos- ible that BST �1- and �2-adrenoceptors have similar or different roles depending on the stimulus. BST treatment with adrenoceptor antagonists did not affect either MAP or HR baseline values. Therefore, although the present study supports the hypothesis that BST noradrenergic neurotransmission plays an impor- tant role in modulating the cardiovascular responses to dynamic exercise, this neurotransmission is not involved in the tonic maintenance of either arterial pressure or HR. These results corroborate previous data in the lit- erature indicating no changes in cardiovascular param- eters after blockade of either glutamatergic, cholinergic or adrenergic receptors in the BST (Alves et al., 2007; Hatam and Nasimi, 2007; Crestani et al., 2008a, 2009a). e t s F. H. F. Alves et al. / Neuroscience 177 (2011) 74–8382 CONCLUSION In conclusion, the present results show that noradrenergic neurotransmission in the BST modulates cardiovascular ad- justments during dynamic exercise in a complex way. Our data provide evidence of an inhibitory influence of BST �1- adrenoceptors on exercise-evoked HR response. 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(Accepted 3 January 2011) (Available online 8 January 2011) Bed nucleus of the stria terminalis 1- and 2-adrenoceptors differentially modulate the cardiovascular responses to exercise in rats Experimental procedures Ethical approval and animals Surgical preparation Measurement of cardiovascular responses Drug microinjection into the BST Experimental protocol Effect of ACSF, WB4101, RX821002 or propranolol microinjection into the BST on dynamic exercise-induced cardiovascular changes Effect of microinjection into the BST of ACSF, WB4101 or RX821002 in the open-field test Histological determination of the microinjection sites Drugs Statistical analysis Results Determination of microinjection sites into the bed nucleus of the stria terminalis Effect of microinjection into the BST of ACSF, WB4101, RX821002 or propranolol on dynamic exercise-induced cardiovascular changes ACSF WB4101 RX821002 Propranolol Effect of microinjection into the BST of ACSF, WB4101 or RX821002 in the open-field test Discussion Conclusion Acknowledgments References