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Estrogen alters the bradycardia response to hypocretin-1 in the nucleus tractus solitarius of the ovariectomized female
Brain Research 978 (2003) 14–23 www.elsevier.com / locate / brainres
Research report
Estrogen alters the bradycardia response to hypocretin-1 in the nucleus tractus solitarius of the ovariectomized female Cleusa V.R. de Oliveira, M. Patricia Rosas-Arellano, L. Pastor Solano-Flores, Tanja Babic, Zhaohui Li, John Ciriello* Department of Physiology, Faculty of Medicine and Dentistry, Health Sciences Centre, University of Western Ontario, London, ON, Canada N6 A 5 C1 Accepted 27 March 2003
Abstract Experiments were performed to investigate the effect of 17b-estradiol (E; 30 pg / ml plasma) treatment (15–25 days) in the ovariectomized (OVX) female Wistar rat on the cardiovascular responses to hypocretin-1 (hcrt-1) in the nucleus tractus solitarius (NTS). In an initial series of experiments, the distribution of hcrt-1-like immunoreactivity within the region of the NTS was mapped in both OVX only and OVX1E animals. Hcrt-1 immunoreactivity was found throughout the NTS region in both groups of females, predominantly within the caudal interstitial, commissural, medial and lateral subnuclei of the NTS. The relative density of hcrt-1 immunoreactivity in all NTS subnuclei was similar in both female groups. Microinjections of hcrt-1 (0.5–10 pmol) into the caudal lateral and medial subnuclei of the NTS complex of the a-chloralose of the urethane-anaesthetized E-treated OVX rat elicited a dose-related decrease in heart rate (HR). On the other hand, although a dose–response effect on arterial pressure was evident, significant arterial pressure responses were observed only at the higher dose of hcrt-1 (.2.5 pmol). In the OVX only female rat, microinjection of hcrt-1 into similar NTS sites elicited a bradycardia and depressor response only at the highest dose of hcrt-1, and these responses were significantly smaller in magnitude than those elicited in the OVX1E animal. In addition, in the OVX only animals, a few sites within the caudal commissural subnucleus of the NTS complex were found at which hcrt-1 elicited tachycardia and pressor responses. Finally, it was found that the reflex bradycardia to the activation of arterial baroreceptors as a result of increasing systemic arterial pressure with phenylephrine (2–4 mg / kg) was significantly potentiated in the OVX1E animals only. These data suggest that hcrt-1 in the NTS of the female activates a neuronal circuit that controls the circulation and that the circulating level of E alters the sensitivity of these cardiovascular circuits to hcrt-1. 2003 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Orexin-A; Blood pressure; Heart rate; Brainstem; Baroreceptor reflex
1. Introduction The newly discovered hypothalamic neuropeptides hypocretin-1 (hcrt-1) and hcrt-2 have been implicated in a variety of homeostatic mechanisms [13,34,35,47]. Hcrt-1 is a 33 amino acid peptide, and hcrt-2 is a 28 amino acid peptide which shares 46% sequence identity with hcrt-1 [35]. These neuropeptides, derived from the same long prepro-orexin molecule by proteolytic cleavage [35], bind *Corresponding author. Tel.: 11-519-661-3484; fax: 11-519-6613827. E-mail address: [email protected] (J. Ciriello).
and activate G protein-coupled receptors [13,35]. Central injections of hcrt-1 have been shown to influence feeding [14,19] and drinking [24] behaviours, sleep and wakefulness [17,44], pain modulation [2], neuroendocrine control [22,46,47] and temperature regulation [49]. In addition, hcrt has been suggested to play an important role in the control of the autonomic nervous system [10,29,39–41]. Intracerebroventricular injections of hcrt-1 have been reported to elicit increases in renal sympathetic activity and catecholamine release, and a long-lasting increase in mean arterial pressure (MAP) [28]. In addition, intracisternal injections of hcrt-1 have been shown to elicit a dosedependent increase in MAP and heart rate (HR) [4], effects
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02724-0
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suggested to be mediated by the activation of sympathetic premotor neurons in the rostral ventrolateral medulla [6,15]. Direct injections of hcrt-1 into this medullary region elicited an increase in AP and HR [4]. It has also been shown that intrathecal injections of hcrt-1 into the intermediolateral nucleus of the thoracolumbar cord elicit increases in MAP and HR, effects suggested to be mediated by activation of preganglionic sympathetic neurons [1]. It has been reported that the nucleus tractus solitarius (NTS), the primary site of termination of cardiovascular afferent fibers [7], receives a direct projection from hcrt-1containing neurons within the cardiovascular region [16] of the lateral hypothalamus [18]. Additionally, intraventricular injections of hcrt-1 have been shown to induce c-fos expression in the NTS [10]. Recently, injections of hcrt-1 into the commissural nucleus of the NTS have been shown to elicit an increase in AP and HR [42]. On the other hand, microinjection of hcrt-1 into the caudal lateral and medial NTS region, where baroreceptor afferent fibers are known to terminate [7], elicited both depressor and bradycardia responses [11] and potentiate the vagal component of the baroreceptor reflex [11]. Epidemiological studies have shown that cardiovascular disease rarely affects women before menopause, suggesting that estrogen (17b-estradiol; E) deficiency may contribute to the etiology of cardiovascular disease in postmenopausal women [8,20]. Experimental studies have also demonstrated that E plays an important role in the maintenance and reflex regulation of autonomic tone [32,36–38]. The NTS is known to contain E receptors [25]. In addition, microinjection of E into the NTS has been shown to decrease renal sympathetic nerve activity, and potentiate the baroreceptor reflex in the female [32,36–38]. Therefore, as both microinjections of hcrt-1 and E into the NTS have been shown to potentiate the baroreflex, the possibility exists that circulating levels of E may exert an effect on NTS circuits that are activated by hcrt-1 in the female. To test this possibility, experiments were performed to investigate the effect of microinjection of hcrt-1 on the MAP, HR and baroreceptor reflex in an anaesthetized, ovariectomized (OVX) female rat that had either been treated with E or OVX only. In addition, as a mapping of hcrt-1 immunoreactivity within the female NTS region is not available, an initial series of experiments was carried out to investigate the distribution of hcrt-1 immunoreactivity in both OVX only and OVX1E animals.
2. Methods and materials Experiments were performed in adult female Wistar rats (250–300 g; Charles River Canada Inc., St. Constant, Canada), either OVX (n516) or OVX and treated with E (11,3,5 [10]-Estratienne-3,17b-diol, Sigma, St Louis, MO, USA) (OVX1E; n516) for 15 to 25 days. The OVX rats
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were implanted immediately after surgery with a silastic capsule (Dow Corning, Midland, MI, USA; internal diameter 1.57 mm, outer diameter 3.175 mm, length 5.0 mm) containing either cholesterol only (ICN Pharmaceutical Inc., Cleveland, OH, USA) for the OVX only rats or a mixture of crystalline E in cholesterol (one part E / three parts cholesterol). These E-containing capsules have previously been shown, using radioimmunoassay (RIA) [12], and confirmed by us using an RIA for E (Ciriello and Solano-Flores, 1999, unpublished observations), to result in a circulating level of approximately 30 pg / ml plasma of E. On the other hand, E levels are not detectable (,1 pg / ml plasma) in OVX animals implanted with a cholesterol capsule after about 3 days [12,27]. The animals received post-operative care and were returned to their home cages. All animals were housed under controlled conditions with a 12 h light / dark cycle. Food and water were available to all animals ad libitum. All experimental procedures were performed in accordance with the guidelines on the use and care of laboratory animals as set by the Canadian Council on Animal Care and approved by the Animal Care Committee at The University of Western Ontario.
2.1. Immunohistochemistry Four animals from the OVX only group and four animals from the OVX1E group under sodium pentobarbital anaesthesia (65 mg / kg i.p.; MTC Pharmaceuticals, Cambridge, ON, Canada) were perfused transcardially with 500 ml of 0.9% physiological saline followed by 500 ml of Zamboni’s fixative containing 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.2–7.4 and 15% saturated picric acid at 4 8C. The brains were removed and stored in a 10% sucrose–PBS solution overnight. Frozen, serial transverse sections of the brainstem at 50 mm were cut in a cryostat (217 8C; Bright model 5030, Bright Instrument Co. Ltd., Huntington, UK). For each animal, one in every two sections of the brainstem was processed immunohistochemically as previously described [5,11] for hcrt-1 immunoreactivity. Brainstem sections were placed in normal goat serum (Vector Laboratories, Burlingame, CA, USA) diluted 1:50 with PBS containing 0.3% Triton X-100 for 30 min. The sections were then rinsed in PBS and placed in primary antisera to hcrt-1 (affinity purified rabbit polyclonal anti-orexin-A; Alpha Diagnostic Intl. Inc., San Antonio, TX, USA; Cat [OXA11-A, Lot [305960A) diluted 1:2000 in PBS / 0.3% Triton X-100 at 4 8C. After 72 h the sections were rinsed in PBS and placed for 30 min in goat biotinylated anti-rabbit IgG (Vector Laboratories) diluted 1:500 in PBS / 0.3% Triton X-100. After a rinse in PBS, the sections were placed in a solution of methanol and hydrogen peroxide (29:1) for 30 min. The sections were then rinsed in PBS and placed in an avidin–biotin complex reagent (Vectastain ABC Elite Kit) in PBS / 0.3% Triton X-100 for 75 min and then washed again in acetate
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buffer at pH 5.5. The peroxidase contained in the ABC reagent was visualized by placing the sections in a solution of 0.006% hydrogen peroxide and 0.02% 3,39-diaminobenzidine tetrahydrochloride (DAB; Sigma) in PBS for 20 min, or in 0.05% DAB, 0.05% hydrogen peroxide and 0.01% nickel ammonium sulfate in acetate buffer for 15–20 min. After rinsing the tissue in PBS, sections were mounted onto gelatinized glass slides, dried and coverglassed. Brainstem sections adjacent to those processed for hcrt-1 immunoreactivity were stained with either Neutral red or thionine for the identification of cytoachitectonic boundaries. The analysis was carried out using bright- and dark-field microscopy. The location of hcrt-1-like-immunoreactive fibers was mapped onto camera lucida projection drawings of the dorsomedial medulla for each experimental case. Controls for hcrt-1-like immunoreactivity included placing brainstem sections in primary hcrt-1 antisera that had been preadsorbed with an excess of hcrt-1 peptide (Cat. [OXA11-P, Alpha Diagnostic), or sections in which the reaction of the tissue with the primary antisera was omitted [5,11]. Under these conditions, no hcrt-1-like immunoreactivity was demonstrated in the brainstem sections.
2.2. Microinjection of hcrt-1 into the NTS On the day of the experiments, the animals were anaesthetized with a-chloralose (60 mg / kg i.v. initially, and supplemented by additional doses of 10–20 mg / kg given every 1–2 h) after induction with equithesin (0.3 ml / 100 g, i.p.). In addition, to determine whether the type of anaesthetic used altered the cardiovascular responses qualitatively or quantitatively [3], experiments were also performed in animals anaesthetized with urethane (1.5 g / kg, i.p.). The trachea was cannulated and the animals were artificially ventilated using a small rodent ventilator (Harvard Apparatus; model 683) with a mixture of room air and 95% O 2 . Body temperature was maintained at 36–37 8C by a heating pad (model K-20-C; American Hospital Supply Corp., Cincinnati, OH, USA). Polyethylene catheters (PE-50; Clay Adams, Parsippany, NJ, USA) were inserted into the femoral artery and vein for the recording of the arterial pressure and the administration of drugs, respectively. Arterial pressure was recorded via a Statham pressure transducer (model P23 XL), and a Grass tachograph (model 7P4K) triggered by the arterial pressure pulse was used to monitor HR. Arterial pressure, MAP and HR were recorded continuously on a Grass polygraph (model 79G). The head of the animal was placed in a Kopf stereotaxic frame and bent downwards at a 458 angle to the horizontal meridian. The dorsal surface of the medulla was exposed by partial occipital craniotomy. The dura was cut and reflected laterally, and the caudal floor of the fourth ventricle was exposed by gently removing the vermis of
the cerebellum by suction. The nervous tissue was kept moist by physiological saline throughout the experiment. Glass micropipettes (tip diameter, 20–35 mm) were pulled from 5 ml Socorex capillary tubing (Mississauga, ON, Canada). Micropipettes were placed stereotaxically into the caudal lateral and medial subnuclei of the NTS complex [10] using obex as a reference point. The microinjection of hcrt-1 (0.5, 1.0, 2.5 and 10 pmol; Phoenix Pharmaceuticals, Mountain View, CA, USA) in 0.9% saline into the NTS was done by the application of pressurized nitrogen pulses controlled by a picospritzer (General Valve, Fairfield, NJ, USA). The injected volume (10–20 nl) was measured by direct observation of the fluid meniscus in the micropipette using a microscope fitted with an ocular micrometer that allowed 2 nl resolution. Two to three sites on each side of the NTS in each animal were tested for the effects of hcrt-1 on MAP and HR. Control injections of the vehicle saline (20 nl) into similar sites of the NTS were shown not to elicit cardiovascular responses. To determine whether hcrt-1 exerted an effect on the baroreceptor reflex, the effect of a microinjection of hcrt-1 (2.5 pmol) into the NTS on the reflex bradycardia elicited by the increase in MAP to an intravenous injection of phenylephrine (PE; 2, 3 or 4 mg / kg) was tested in animals under urethane anaesthesia [9]. Injections of PE were made 5 min before (control) and 0.5, 2.5 and 5.0 min after microinjection of hcrt-1 into the NTS. In each animal, injections of 2, 3 or 4 mg / kg PE (in 0.05–0.1 ml of saline) were made while testing only one site in each side of the NTS. At the end of all experiments, the micropipette was withdrawn from the last site of hcrt-1 microinjection, emptied of hcrt-1 solution, filled with Pontamine Sky blue in 0.9% saline and lowered back stereotaxically into the same site at which a 20 nl microinjection of the dye was made to mark the injection site in the NTS. The animals were perfused with 50 ml of 0.9% saline solution followed by 50 ml of 10% formalin. Injections of the Pontamine Sky blue dye did not elicit cardiovascular responses at the site at which the hcrt-1 previously elicited a depressor and bradycardia response. The brains were post-fixed in 10% buffered formalin solution for 2–4 days. Frozen, transverse sections of the brainstem were cut in a cryostat at 50 mm, mounted on glass slides, and stained with Neutral red. Stimulation sites were determined by extrapolation along the pipette tract from the centre of the marked injection site. All stimulation sites were mapped on projection drawings of transverse sections of the rat brainstem for each animal and later plotted on a standard set drawing of sections of the dorsomedial medulla modified from a rat stereotaxic atlas [33]. Means6standard error of the means were calculated for the magnitude of the peak changes in MAP and HR and the duration of the responses. A response was defined as a change in MAP or HR of greater than 5 mmHg or 10 bpm,
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respectively. Comparisons of the changes in MAP or HR between OVX only and OVX1E groups were made using an analysis of variance for repeated measures followed by a Bonferroni post-hoc test. The effect of hcrt-1 injections on the baroreceptor reflex was analysed using a regression analysis and statistical comparisons among the slopes of the lines were made using an analysis of variance followed by Dunnett’s multiple comparison test. In all statistical comparisons, P,0.05 was taken to indicate statistical significance.
3. Results
3.1. Hcrt-1 immunoreactivity within the NTS region Fig. 1 summarizes the distribution of hcrt-1-like immunoreactivity in the NTS of the female. The distribution and density of hcrt-1 fibers in the dorsomedial medulla of the OVX1E female (Figs. 1 and 2) did not differ from that of the OVX only female. In the female, hcrt-1-labelled axon and axon terminals were observed throughout the NTS complex. Light to moderate hcrt-1 immunoreactivity was found within the dorsal lateral (Fig. 2a), medial and
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commissural subnuclei of the NTS. Additionally, relatively moderate to dense labelling was found within the interstitial subnucleus of the NTS (Fig. 2b) compared to other subnuclei in the NTS. Of note was the apparent lack of hcrt-1 immunoreactivity within the central subnucleus of the NTS. Within medullary structures adjacent to NTS that comprise the dorsal vagal complex, it was interesting to note that the dorsal motor nucleus of the vagus (DMV) contained very few, if any, labelled fibers. The few fibers that were observed within the region of the DMV appeared to course through and around the lateral edges of the nucleus en route to the NTS. Similarly, the area postrema contained only a few hcrt-1-labelled fibers.
3.2. Cardiovascular effects of microinjection of hcrt-1 into the NTS To determine the effect of hcrt-1 on the MAP and HR, hcrt-1 was microinjected at three different dosages at histologically verified sites in the caudal dorsolateral and medial subnuclei of the NTS complex (Fig. 3). Baseline MAP in the anaesthetized OVX only female (112.964.1 mmHg) was found to be different from that observed in the OVX1E female (93.665.4 mmHg) under anaesthesia.
Fig. 1. A series of camera lucida projection drawings through the caudal dorsomedial medulla of the rat extending from 5.3 to 3.8 mm caudal to the interaural line showing the distribution of hcrt-1-like immunoreactivity in the OVX1E female rat. Note that labelling of fibers was observed bilaterally, although only one side is presented. In addition, note that the distribution of hcrt-1 immunoreactivity was not different in either the OVX only or OVX1E animals. AP, area postrema; DMV, dorsal motor nucleus of the vagus; cc, central canal; Com, commissural subnucleus of nucleus of the solitary tract (NTS); Cu, nucleus cuneatus; fg, fasiculus gracilis; Gr, nucleus gracilis; Mv, medial vestibular nucleus; PCRt, parvocellular reticular nucleus; Sc, central subnucleus of NTS; Sg, subnucleus gelatinosa of NTS; Slt, lateral subnucleus of NTS; Sm, medial subnucleus of NTS; Sni, interstitial subnucleus of NTS; St, tractus solitarius; Sv, ventral subnucleus of NTS; 4V, 4th ventricle; 12 M, hypoglossal nucleus. Calibration mark, 500 mm.
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Fig. 2. Bright-field photomicrographs showing hcrt-1-like immunoreactivity in the lateral (Slt; a) and interstitial (Sni; b) subnuclei of the NTS complex in the OVX1E female rat. Note that, within the Sni, presumptive hcrt-1 terminal labelling (arrow, punctate reaction product) was present. DMV, dorsal motor nucleus of the vagus. Refer to Fig. 1 for additional abbreviations. Calibration mark in (b) represents 100 mm and also applies to (a).
Although the resting HR in both group of animals was similar, the HR in the OVX1E (355.7623.8 bpm) tended to be lower than in the OVX only animals (379.968.4 bpm). Microinjection of hcrt-1 into the dorsolateral and medial subnuclei of the NTS complex of OVX1E rats elicited a dose-related decrease in HR and MAP (Fig. 4). On the other hand, significant decreases in MAP and HR were elicited in the OVX only animals at only the higher dose of
hcrt-1 (Figs. 4 and 5). The magnitude of the HR responses was greater in the OVX1E group at all doses of hcrt-1 microinjected compared with the OVX only group. Although at the higher dosages (.2.5 pmol) of hcrt-1 there was an apparent trend in the magnitude of the MAP depressor responses to be greater in the OVX1E rat, the difference was not statistically significant (Figs. 4 and 5). However, as shown in the representative responses in MAP and HR elicited by a microinjection of 2.5 pmol of
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Fig. 3. A series of transverse sections of the dorsal medial medulla modified from a stereotaxic atlas [33] extending from 4.3 to 5.1 mm caudal to the interaural line showing the location of histologically verified sites corresponding to the center of hcrt-1 microinjections (2.5 pmol) into the NTS complex of the OVX1E (left) and OVX (right) rat. (d) Sites eliciting decreases in MAP and / or HR, ( ) sites at which hcrt-1 microinjections did not elicit cardiovascular responses, and (m) sites that elicited increases in HR and / or MAP. Note that not all sites injected are shown as many overlapped those indicated on the sections. In addition, note that increases in HR and / or MAP were only observed in the caudal commissural nucleus of the NTS complex of the OVX only animals. AP, area postrema; 12 M, hypoglossal nucleus; 4v, 4th ventricle; cc, central canal; Com, commissural subnucleus of the NTS; DMV, dorsal motor nucleus of the vagus; cc, central canal; Slt, dorsolateral subnucleus of the NTS; Sm, medial subnucleus of the NTS; St, tractus solitarius; GR, nucleus gracilis; Cu, cuneate nucleus; Ecu, external cuneate nucleus. Calibration mark, 500 mm.
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hcrt-1 into a site in the dorsolateral NTS in an OVX1E rat (Fig. 5a) and at a corresponding site in the OVX only animal (Fig. 5b), the durations of both the depressor and bradycardia responses elicited in the OVX1E animals (OVX1E, MAP duration 211.1644.6 s, HR duration 472.0645.1 s; OVX only, MAP duration 144.3628.5 s, HR duration 192.5651.2 s) were significantly longer. In the OVX only animals, microinjections of the higher doses of hcrt-1 (2.5 pmol) into a few sites in the caudal medial and commissural subnuclei of the NTS complex
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Fig. 4. Bar chart showing the effect of microinjection of varying amounts of hcrt-1 (0.5–10 pmol) into the dorsolateral and medial subnuclei of the NTS on the magnitude of the mean arterial pressure (MAP; top panel) and heart rate (HR; bottom panel) response in OVX1E (hatched bars) and OVX only (open bars) female rats. All values are means6S.E. of the mean. Numbers in parentheses indicate number of NTS sites injected at that specific dose of hcrt-1. Note that the magnitude of the bradycardia response at all dosages was significantly greater (*P,0.05) in OVX1E animals than in OVX animals.
were found to elicit increases in MAP (n57; 11.462.6 mmHg) and / or HR (n52; 15.065.0 bpm) (Fig. 3). Similar responses were occasionally observed with injections of a 10 pmol dose of hcrt-1 into the commissural nucleus. Injection of hcrt-1 into areas immediately outside the NTS complex in both the OVX1E and the OVX only rats did not elicit any cardiovascular response (Fig. 3). In addition, injections of similar volumes of saline, the vehicle, into these NTS sites did not elicit cardiovascular responses. The effect of hcrt-1 microinjection into the dorsolateral or medial subnuclei of the NTS on the reflex bradycardia to activation of arterial baroreceptors following an acute rise in systemic AP was investigated in the OVX only and the OVX1E anaesthetized female rat (Fig. 6). Activation of NTS neurons by hcrt-1 significantly potentiated the reflex decrease in HR to baroreceptor activation in the OVX1E animals (Fig. 6, top panel). On the other hand, no effect was observed on the reflex bradycardia in the OVX only rats (Fig. 6, bottom panel).
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Fig. 5. Representative heart rate (HR), arterial pressure (AP), and mean AP (MAP) responses to a microinjection of 2.5 pmol of hcrt-1 (arrows) into the caudal dorsolateral NTS in OVX1E (a) and OVX only (b) rats. Note that the HR response is both of greater amplitude and duration in the OVX1E animal compared to the OVX only rat. On the other hand, although the magnitude of the depressor response in both the OVX1E and OVX only animals appears to be the same, the duration of the response is longer in the OVX1E animal. Calibration mark, 1 min.
4. Discussion Hcrt-containing neurons, located exclusively within the lateral hypothalamus [13,30,47,50], have been shown to extensively innervate different levels of the neuroaxis [18,30,35,47,48], including the NTS complex in the male rat [10,18,30]. The NTS is an area well known to be involved in reflex regulation of cardiac rate and vasomotor tone [7]. This study has demonstrated that injections of hcrt-1 into the NTS of the female rat elicited dose-related bradycardia and depressor responses that were greater in magnitude and / or duration in the OVX1E group compared with the OVX only animals. In addition, the data show that hcrt-1 in the NTS potentiated the reflex bradycardia to activation of the arterial baroreflex in the OVX1E rats only, suggesting that the neuronal circuits involved in these cardiovascular responses to activation of hcrt-1 are modulated by the circulating levels of E. The distribution and density of hcrt-1 immunoreactivity within the NTS complex was not altered by the circulating level of E in the OVX female. In both OVX only and in OVX1E-treated female rats, hcrt-1-labelled axons and axon terminals were found to innervate similar subnuclei of the NTS complex. However, it is interesting to note that by comparing Fig. 1 in the report by de Oliveira et al. [11] with Fig. 1 presented in this study on the distribution of hcrt-1 immunoreactivity within the NTS complex, it is readily apparent that, in the male, both the distribution and density of hcrt-1 labelling is different compared to the females. In the female, all structures in the NTS complex appear to receive a less dense innervation by hcrt-1 axons. In addition, the dorsal motor nucleus of the vagus was
Fig. 6. Graph showing the effect of hcrt-1 microinjection into the dorsolateral subnucleus of the nucleus of the solitary tract complex on the reflex bradycardia elicited by activation of arterial baroreceptors following an acute increase in mean arterial pressure (MAP) resulting from an i.v. injection of phenylephrine in OVX1E animals (top graph) and OVX only animals (bottom graph). Note that the HR component of the baroreceptor reflex is significantly (P,0.05) increased only in the OVX1 E rats at about 0.5 min (P,0.05) after the hcrt-1 injection. The reflex HR response begins to recover towards control values by 2.5 min. n, number of responses used to calculate each point plotted on the graph. All values are means6S.E.M. Control (d; 5 min before hcrt-1 injection; OVX1E, R 2 5 0.8194; OVX only, R 2 5 0.9945). Time after hcrt-1 injections: 0.5 min (m; OVX1E, R 2 5 0.8888; OVX, R 2 5 0.8707), and 2.5 min (j; OVX1E, R 2 5 0.9966; OVX only, R 2 5 0.0.7937).
innervated by hcrt-1-labelled axons in the male, while hcrt-1 labelling within this structure in the female was not apparent. In support of this latter observation, recently, in the male rat, hcrt-1 has been shown to evoke gastric acid secretion [45] and to directly excite the dorsal motor nucleus of the vagus motor neurons in vitro [21]. The dorsal motor nucleus of the vagus is known to be involved in the parasympathetic regulation of gastrointestinal function [23]. These data suggest not only a gender difference
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in the innervation of the dorsal vagal complex, but also in the function of hcrt-1 in this area between the female and male. The finding that activation of the dorsolateral and medial subnuclei of the NTS complex, regions that have previously been shown to receive baroreceptor afferent projections [7], elicits bradycardia and depressor responses and alters the baroreflex was not unexpected, as these cardiovascular responses elicited by hcrt-1 into the NTS are consistent with those observed during electrical or glutamate stimulation of this NTS region, and during activation of the baroreceptor reflex [7,26]. In addition, the responses to activation of the NTS neurons by hcrt-1 in the female rat are qualitatively similar to those reported in an earlier study in the male rat in which hcrt-1 injection into the dorsolateral and medial subnuclei of the NTS complex elicited bradycardia and depressor responses [11]. However, it is interesting to note in this study that circulating E within physiological levels (approximately 30 pg / ml plasma) affected the magnitude of these cardiovascular responses. The HR and MAP responses were greater in magnitude in OVX1E animals compared to OVX only animals. In addition, the responses in the OVX1E animals appear to be smaller in magnitude than those previously reported in the male rat [11]. Although a direct comparison between the magnitude of the cardiovascular responses in the two different sexes was not made, it is evident that the responses elicited in the male [11] were generally about 30% greater than in the OVX1E female. This finding is consistent with our observation that the density of hcrt-1 immunoreactivity in the NTS complex of either the OVX only or OVX1E female is considerably less than that in the male [11]. Taken together, these data suggest not only that a gender difference exists between the functional innervation of the NTS by hcrt-1 pathways, but also that the levels of plasma E influence the sensitivity of neurons involved in the reflex control of the circulation to hcrt-1. In addition, these data suggest that the differences observed in this study between OVX only and OVX1E females in the magnitude of the cardiovascular responses to hcrt-1 is likely not due to the innervation of the NTS by hcrt-1 axons, but may reflect changes in the expression of hcrt-1 receptors on NTS neurons in the OVX1E female rat. Although the mechanism by which E may alter the neuronal circuits in the NTS to potentiate the bradycardia response to hcrt-1 in the OVX1E animals is not known, it has been demonstrated that neurons within the caudal NTS complex contain E receptors [25], and injections of E into the NTS complex produce a decrease in resting arterial pressure and potentiate the reflex bradycardia to activation of arterial baroreceptors [36–38]. In addition, it has been reported that E treatment in the OVX mouse can facilitate the baroreceptor reflex [32]. Furthermore, it has been shown that E can alter the release of glutamate [28], a finding that has also been suggested as a mechanism of action for hcrt-1 in the hypothalamus [44] and in NTS
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neurons [43]. These data would therefore suggest that hcrt-1 may act to either presynaptically increase the release of glutamate [43,44], the putative neurotransmitter in baroreceptor afferent fibers [31], or to interact with E sensitive interneurons in the NTS complex that facilitate the baroreflex. It has been reported that the bradycardia response elicited by hcrt-1 microinjection into the NTS in the male rat is mediated predominantly by the activation of central vagal cardiomotor neurons [11]. E has been shown to facilitate parasympathetic activity to the heart by potentiating the effect of baroreceptor afferent inputs into the nucleus ambiguus [36–38]. Therefore, the finding in this study that the HR response to activation of the baroreflex, which is mainly due to vagal activation [9], during hcrt-1 injections was potentiated only in the E-treated animals suggests that hcrt-1 acts on central NTS circuits that are also modified by E. It was also observed that, in the OVX only animal, a small number of sites located in the commissural subnucleus of the NTS elicited tachycardia and pressor responses, an observation supported by an earlier study that identified pressor sites to hcrt-1 in the commissural nucleus of NTS [11,40]. The caudal commissural nucleus of the NTS complex has been shown to receive mainly chemoreceptor afferent inputs [7]. Taken together, these data suggest that hcrt-1 may be involved in the activation of neuronal systems in the NTS region that may mediate sympathoexcitatory responses to chemoreceptor activation. However, it should be noted that pressor sites to hcrt-1 injections were not found within the NTS complex of OVX1E animals. Therefore, these data suggest that the circulating level of E may act to inhibit neuronal systems in the NTS activated by hcrt-1 that increase sympathetic nervous system activity. In summary, data have been presented indicating that microinjections of hcrt-1 into the NTS elicit bradycardia and arterial pressure depressor responses that are greater in the OVX female treated with E compared to the non-Etreated OVX female. In addition, it has been shown that hcrt-1 potentiates the baroreceptor reflex in these E-treated females. Taken together, the data suggest that E alters the sensitivity of NTS mechanisms involved in the reflex regulation of the circulation to hcrt-1.
Acknowledgements This work was supported by a research grant from the Heart and Stroke Foundation of Ontario to Dr. J. Ciriello.
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Research report
Estrogen alters the bradycardia response to hypocretin-1 in the nucleus tractus solitarius of the ovariectomized female Cleusa V.R. de Oliveira, M. Patricia Rosas-Arellano, L. Pastor Solano-Flores, Tanja Babic, Zhaohui Li, John Ciriello* Department of Physiology, Faculty of Medicine and Dentistry, Health Sciences Centre, University of Western Ontario, London, ON, Canada N6 A 5 C1 Accepted 27 March 2003
Abstract Experiments were performed to investigate the effect of 17b-estradiol (E; 30 pg / ml plasma) treatment (15–25 days) in the ovariectomized (OVX) female Wistar rat on the cardiovascular responses to hypocretin-1 (hcrt-1) in the nucleus tractus solitarius (NTS). In an initial series of experiments, the distribution of hcrt-1-like immunoreactivity within the region of the NTS was mapped in both OVX only and OVX1E animals. Hcrt-1 immunoreactivity was found throughout the NTS region in both groups of females, predominantly within the caudal interstitial, commissural, medial and lateral subnuclei of the NTS. The relative density of hcrt-1 immunoreactivity in all NTS subnuclei was similar in both female groups. Microinjections of hcrt-1 (0.5–10 pmol) into the caudal lateral and medial subnuclei of the NTS complex of the a-chloralose of the urethane-anaesthetized E-treated OVX rat elicited a dose-related decrease in heart rate (HR). On the other hand, although a dose–response effect on arterial pressure was evident, significant arterial pressure responses were observed only at the higher dose of hcrt-1 (.2.5 pmol). In the OVX only female rat, microinjection of hcrt-1 into similar NTS sites elicited a bradycardia and depressor response only at the highest dose of hcrt-1, and these responses were significantly smaller in magnitude than those elicited in the OVX1E animal. In addition, in the OVX only animals, a few sites within the caudal commissural subnucleus of the NTS complex were found at which hcrt-1 elicited tachycardia and pressor responses. Finally, it was found that the reflex bradycardia to the activation of arterial baroreceptors as a result of increasing systemic arterial pressure with phenylephrine (2–4 mg / kg) was significantly potentiated in the OVX1E animals only. These data suggest that hcrt-1 in the NTS of the female activates a neuronal circuit that controls the circulation and that the circulating level of E alters the sensitivity of these cardiovascular circuits to hcrt-1. 2003 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Orexin-A; Blood pressure; Heart rate; Brainstem; Baroreceptor reflex
1. Introduction The newly discovered hypothalamic neuropeptides hypocretin-1 (hcrt-1) and hcrt-2 have been implicated in a variety of homeostatic mechanisms [13,34,35,47]. Hcrt-1 is a 33 amino acid peptide, and hcrt-2 is a 28 amino acid peptide which shares 46% sequence identity with hcrt-1 [35]. These neuropeptides, derived from the same long prepro-orexin molecule by proteolytic cleavage [35], bind *Corresponding author. Tel.: 11-519-661-3484; fax: 11-519-6613827. E-mail address: [email protected] (J. Ciriello).
and activate G protein-coupled receptors [13,35]. Central injections of hcrt-1 have been shown to influence feeding [14,19] and drinking [24] behaviours, sleep and wakefulness [17,44], pain modulation [2], neuroendocrine control [22,46,47] and temperature regulation [49]. In addition, hcrt has been suggested to play an important role in the control of the autonomic nervous system [10,29,39–41]. Intracerebroventricular injections of hcrt-1 have been reported to elicit increases in renal sympathetic activity and catecholamine release, and a long-lasting increase in mean arterial pressure (MAP) [28]. In addition, intracisternal injections of hcrt-1 have been shown to elicit a dosedependent increase in MAP and heart rate (HR) [4], effects
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02724-0
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suggested to be mediated by the activation of sympathetic premotor neurons in the rostral ventrolateral medulla [6,15]. Direct injections of hcrt-1 into this medullary region elicited an increase in AP and HR [4]. It has also been shown that intrathecal injections of hcrt-1 into the intermediolateral nucleus of the thoracolumbar cord elicit increases in MAP and HR, effects suggested to be mediated by activation of preganglionic sympathetic neurons [1]. It has been reported that the nucleus tractus solitarius (NTS), the primary site of termination of cardiovascular afferent fibers [7], receives a direct projection from hcrt-1containing neurons within the cardiovascular region [16] of the lateral hypothalamus [18]. Additionally, intraventricular injections of hcrt-1 have been shown to induce c-fos expression in the NTS [10]. Recently, injections of hcrt-1 into the commissural nucleus of the NTS have been shown to elicit an increase in AP and HR [42]. On the other hand, microinjection of hcrt-1 into the caudal lateral and medial NTS region, where baroreceptor afferent fibers are known to terminate [7], elicited both depressor and bradycardia responses [11] and potentiate the vagal component of the baroreceptor reflex [11]. Epidemiological studies have shown that cardiovascular disease rarely affects women before menopause, suggesting that estrogen (17b-estradiol; E) deficiency may contribute to the etiology of cardiovascular disease in postmenopausal women [8,20]. Experimental studies have also demonstrated that E plays an important role in the maintenance and reflex regulation of autonomic tone [32,36–38]. The NTS is known to contain E receptors [25]. In addition, microinjection of E into the NTS has been shown to decrease renal sympathetic nerve activity, and potentiate the baroreceptor reflex in the female [32,36–38]. Therefore, as both microinjections of hcrt-1 and E into the NTS have been shown to potentiate the baroreflex, the possibility exists that circulating levels of E may exert an effect on NTS circuits that are activated by hcrt-1 in the female. To test this possibility, experiments were performed to investigate the effect of microinjection of hcrt-1 on the MAP, HR and baroreceptor reflex in an anaesthetized, ovariectomized (OVX) female rat that had either been treated with E or OVX only. In addition, as a mapping of hcrt-1 immunoreactivity within the female NTS region is not available, an initial series of experiments was carried out to investigate the distribution of hcrt-1 immunoreactivity in both OVX only and OVX1E animals.
2. Methods and materials Experiments were performed in adult female Wistar rats (250–300 g; Charles River Canada Inc., St. Constant, Canada), either OVX (n516) or OVX and treated with E (11,3,5 [10]-Estratienne-3,17b-diol, Sigma, St Louis, MO, USA) (OVX1E; n516) for 15 to 25 days. The OVX rats
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were implanted immediately after surgery with a silastic capsule (Dow Corning, Midland, MI, USA; internal diameter 1.57 mm, outer diameter 3.175 mm, length 5.0 mm) containing either cholesterol only (ICN Pharmaceutical Inc., Cleveland, OH, USA) for the OVX only rats or a mixture of crystalline E in cholesterol (one part E / three parts cholesterol). These E-containing capsules have previously been shown, using radioimmunoassay (RIA) [12], and confirmed by us using an RIA for E (Ciriello and Solano-Flores, 1999, unpublished observations), to result in a circulating level of approximately 30 pg / ml plasma of E. On the other hand, E levels are not detectable (,1 pg / ml plasma) in OVX animals implanted with a cholesterol capsule after about 3 days [12,27]. The animals received post-operative care and were returned to their home cages. All animals were housed under controlled conditions with a 12 h light / dark cycle. Food and water were available to all animals ad libitum. All experimental procedures were performed in accordance with the guidelines on the use and care of laboratory animals as set by the Canadian Council on Animal Care and approved by the Animal Care Committee at The University of Western Ontario.
2.1. Immunohistochemistry Four animals from the OVX only group and four animals from the OVX1E group under sodium pentobarbital anaesthesia (65 mg / kg i.p.; MTC Pharmaceuticals, Cambridge, ON, Canada) were perfused transcardially with 500 ml of 0.9% physiological saline followed by 500 ml of Zamboni’s fixative containing 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.2–7.4 and 15% saturated picric acid at 4 8C. The brains were removed and stored in a 10% sucrose–PBS solution overnight. Frozen, serial transverse sections of the brainstem at 50 mm were cut in a cryostat (217 8C; Bright model 5030, Bright Instrument Co. Ltd., Huntington, UK). For each animal, one in every two sections of the brainstem was processed immunohistochemically as previously described [5,11] for hcrt-1 immunoreactivity. Brainstem sections were placed in normal goat serum (Vector Laboratories, Burlingame, CA, USA) diluted 1:50 with PBS containing 0.3% Triton X-100 for 30 min. The sections were then rinsed in PBS and placed in primary antisera to hcrt-1 (affinity purified rabbit polyclonal anti-orexin-A; Alpha Diagnostic Intl. Inc., San Antonio, TX, USA; Cat [OXA11-A, Lot [305960A) diluted 1:2000 in PBS / 0.3% Triton X-100 at 4 8C. After 72 h the sections were rinsed in PBS and placed for 30 min in goat biotinylated anti-rabbit IgG (Vector Laboratories) diluted 1:500 in PBS / 0.3% Triton X-100. After a rinse in PBS, the sections were placed in a solution of methanol and hydrogen peroxide (29:1) for 30 min. The sections were then rinsed in PBS and placed in an avidin–biotin complex reagent (Vectastain ABC Elite Kit) in PBS / 0.3% Triton X-100 for 75 min and then washed again in acetate
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buffer at pH 5.5. The peroxidase contained in the ABC reagent was visualized by placing the sections in a solution of 0.006% hydrogen peroxide and 0.02% 3,39-diaminobenzidine tetrahydrochloride (DAB; Sigma) in PBS for 20 min, or in 0.05% DAB, 0.05% hydrogen peroxide and 0.01% nickel ammonium sulfate in acetate buffer for 15–20 min. After rinsing the tissue in PBS, sections were mounted onto gelatinized glass slides, dried and coverglassed. Brainstem sections adjacent to those processed for hcrt-1 immunoreactivity were stained with either Neutral red or thionine for the identification of cytoachitectonic boundaries. The analysis was carried out using bright- and dark-field microscopy. The location of hcrt-1-like-immunoreactive fibers was mapped onto camera lucida projection drawings of the dorsomedial medulla for each experimental case. Controls for hcrt-1-like immunoreactivity included placing brainstem sections in primary hcrt-1 antisera that had been preadsorbed with an excess of hcrt-1 peptide (Cat. [OXA11-P, Alpha Diagnostic), or sections in which the reaction of the tissue with the primary antisera was omitted [5,11]. Under these conditions, no hcrt-1-like immunoreactivity was demonstrated in the brainstem sections.
2.2. Microinjection of hcrt-1 into the NTS On the day of the experiments, the animals were anaesthetized with a-chloralose (60 mg / kg i.v. initially, and supplemented by additional doses of 10–20 mg / kg given every 1–2 h) after induction with equithesin (0.3 ml / 100 g, i.p.). In addition, to determine whether the type of anaesthetic used altered the cardiovascular responses qualitatively or quantitatively [3], experiments were also performed in animals anaesthetized with urethane (1.5 g / kg, i.p.). The trachea was cannulated and the animals were artificially ventilated using a small rodent ventilator (Harvard Apparatus; model 683) with a mixture of room air and 95% O 2 . Body temperature was maintained at 36–37 8C by a heating pad (model K-20-C; American Hospital Supply Corp., Cincinnati, OH, USA). Polyethylene catheters (PE-50; Clay Adams, Parsippany, NJ, USA) were inserted into the femoral artery and vein for the recording of the arterial pressure and the administration of drugs, respectively. Arterial pressure was recorded via a Statham pressure transducer (model P23 XL), and a Grass tachograph (model 7P4K) triggered by the arterial pressure pulse was used to monitor HR. Arterial pressure, MAP and HR were recorded continuously on a Grass polygraph (model 79G). The head of the animal was placed in a Kopf stereotaxic frame and bent downwards at a 458 angle to the horizontal meridian. The dorsal surface of the medulla was exposed by partial occipital craniotomy. The dura was cut and reflected laterally, and the caudal floor of the fourth ventricle was exposed by gently removing the vermis of
the cerebellum by suction. The nervous tissue was kept moist by physiological saline throughout the experiment. Glass micropipettes (tip diameter, 20–35 mm) were pulled from 5 ml Socorex capillary tubing (Mississauga, ON, Canada). Micropipettes were placed stereotaxically into the caudal lateral and medial subnuclei of the NTS complex [10] using obex as a reference point. The microinjection of hcrt-1 (0.5, 1.0, 2.5 and 10 pmol; Phoenix Pharmaceuticals, Mountain View, CA, USA) in 0.9% saline into the NTS was done by the application of pressurized nitrogen pulses controlled by a picospritzer (General Valve, Fairfield, NJ, USA). The injected volume (10–20 nl) was measured by direct observation of the fluid meniscus in the micropipette using a microscope fitted with an ocular micrometer that allowed 2 nl resolution. Two to three sites on each side of the NTS in each animal were tested for the effects of hcrt-1 on MAP and HR. Control injections of the vehicle saline (20 nl) into similar sites of the NTS were shown not to elicit cardiovascular responses. To determine whether hcrt-1 exerted an effect on the baroreceptor reflex, the effect of a microinjection of hcrt-1 (2.5 pmol) into the NTS on the reflex bradycardia elicited by the increase in MAP to an intravenous injection of phenylephrine (PE; 2, 3 or 4 mg / kg) was tested in animals under urethane anaesthesia [9]. Injections of PE were made 5 min before (control) and 0.5, 2.5 and 5.0 min after microinjection of hcrt-1 into the NTS. In each animal, injections of 2, 3 or 4 mg / kg PE (in 0.05–0.1 ml of saline) were made while testing only one site in each side of the NTS. At the end of all experiments, the micropipette was withdrawn from the last site of hcrt-1 microinjection, emptied of hcrt-1 solution, filled with Pontamine Sky blue in 0.9% saline and lowered back stereotaxically into the same site at which a 20 nl microinjection of the dye was made to mark the injection site in the NTS. The animals were perfused with 50 ml of 0.9% saline solution followed by 50 ml of 10% formalin. Injections of the Pontamine Sky blue dye did not elicit cardiovascular responses at the site at which the hcrt-1 previously elicited a depressor and bradycardia response. The brains were post-fixed in 10% buffered formalin solution for 2–4 days. Frozen, transverse sections of the brainstem were cut in a cryostat at 50 mm, mounted on glass slides, and stained with Neutral red. Stimulation sites were determined by extrapolation along the pipette tract from the centre of the marked injection site. All stimulation sites were mapped on projection drawings of transverse sections of the rat brainstem for each animal and later plotted on a standard set drawing of sections of the dorsomedial medulla modified from a rat stereotaxic atlas [33]. Means6standard error of the means were calculated for the magnitude of the peak changes in MAP and HR and the duration of the responses. A response was defined as a change in MAP or HR of greater than 5 mmHg or 10 bpm,
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respectively. Comparisons of the changes in MAP or HR between OVX only and OVX1E groups were made using an analysis of variance for repeated measures followed by a Bonferroni post-hoc test. The effect of hcrt-1 injections on the baroreceptor reflex was analysed using a regression analysis and statistical comparisons among the slopes of the lines were made using an analysis of variance followed by Dunnett’s multiple comparison test. In all statistical comparisons, P,0.05 was taken to indicate statistical significance.
3. Results
3.1. Hcrt-1 immunoreactivity within the NTS region Fig. 1 summarizes the distribution of hcrt-1-like immunoreactivity in the NTS of the female. The distribution and density of hcrt-1 fibers in the dorsomedial medulla of the OVX1E female (Figs. 1 and 2) did not differ from that of the OVX only female. In the female, hcrt-1-labelled axon and axon terminals were observed throughout the NTS complex. Light to moderate hcrt-1 immunoreactivity was found within the dorsal lateral (Fig. 2a), medial and
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commissural subnuclei of the NTS. Additionally, relatively moderate to dense labelling was found within the interstitial subnucleus of the NTS (Fig. 2b) compared to other subnuclei in the NTS. Of note was the apparent lack of hcrt-1 immunoreactivity within the central subnucleus of the NTS. Within medullary structures adjacent to NTS that comprise the dorsal vagal complex, it was interesting to note that the dorsal motor nucleus of the vagus (DMV) contained very few, if any, labelled fibers. The few fibers that were observed within the region of the DMV appeared to course through and around the lateral edges of the nucleus en route to the NTS. Similarly, the area postrema contained only a few hcrt-1-labelled fibers.
3.2. Cardiovascular effects of microinjection of hcrt-1 into the NTS To determine the effect of hcrt-1 on the MAP and HR, hcrt-1 was microinjected at three different dosages at histologically verified sites in the caudal dorsolateral and medial subnuclei of the NTS complex (Fig. 3). Baseline MAP in the anaesthetized OVX only female (112.964.1 mmHg) was found to be different from that observed in the OVX1E female (93.665.4 mmHg) under anaesthesia.
Fig. 1. A series of camera lucida projection drawings through the caudal dorsomedial medulla of the rat extending from 5.3 to 3.8 mm caudal to the interaural line showing the distribution of hcrt-1-like immunoreactivity in the OVX1E female rat. Note that labelling of fibers was observed bilaterally, although only one side is presented. In addition, note that the distribution of hcrt-1 immunoreactivity was not different in either the OVX only or OVX1E animals. AP, area postrema; DMV, dorsal motor nucleus of the vagus; cc, central canal; Com, commissural subnucleus of nucleus of the solitary tract (NTS); Cu, nucleus cuneatus; fg, fasiculus gracilis; Gr, nucleus gracilis; Mv, medial vestibular nucleus; PCRt, parvocellular reticular nucleus; Sc, central subnucleus of NTS; Sg, subnucleus gelatinosa of NTS; Slt, lateral subnucleus of NTS; Sm, medial subnucleus of NTS; Sni, interstitial subnucleus of NTS; St, tractus solitarius; Sv, ventral subnucleus of NTS; 4V, 4th ventricle; 12 M, hypoglossal nucleus. Calibration mark, 500 mm.
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Fig. 2. Bright-field photomicrographs showing hcrt-1-like immunoreactivity in the lateral (Slt; a) and interstitial (Sni; b) subnuclei of the NTS complex in the OVX1E female rat. Note that, within the Sni, presumptive hcrt-1 terminal labelling (arrow, punctate reaction product) was present. DMV, dorsal motor nucleus of the vagus. Refer to Fig. 1 for additional abbreviations. Calibration mark in (b) represents 100 mm and also applies to (a).
Although the resting HR in both group of animals was similar, the HR in the OVX1E (355.7623.8 bpm) tended to be lower than in the OVX only animals (379.968.4 bpm). Microinjection of hcrt-1 into the dorsolateral and medial subnuclei of the NTS complex of OVX1E rats elicited a dose-related decrease in HR and MAP (Fig. 4). On the other hand, significant decreases in MAP and HR were elicited in the OVX only animals at only the higher dose of
hcrt-1 (Figs. 4 and 5). The magnitude of the HR responses was greater in the OVX1E group at all doses of hcrt-1 microinjected compared with the OVX only group. Although at the higher dosages (.2.5 pmol) of hcrt-1 there was an apparent trend in the magnitude of the MAP depressor responses to be greater in the OVX1E rat, the difference was not statistically significant (Figs. 4 and 5). However, as shown in the representative responses in MAP and HR elicited by a microinjection of 2.5 pmol of
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Fig. 3. A series of transverse sections of the dorsal medial medulla modified from a stereotaxic atlas [33] extending from 4.3 to 5.1 mm caudal to the interaural line showing the location of histologically verified sites corresponding to the center of hcrt-1 microinjections (2.5 pmol) into the NTS complex of the OVX1E (left) and OVX (right) rat. (d) Sites eliciting decreases in MAP and / or HR, ( ) sites at which hcrt-1 microinjections did not elicit cardiovascular responses, and (m) sites that elicited increases in HR and / or MAP. Note that not all sites injected are shown as many overlapped those indicated on the sections. In addition, note that increases in HR and / or MAP were only observed in the caudal commissural nucleus of the NTS complex of the OVX only animals. AP, area postrema; 12 M, hypoglossal nucleus; 4v, 4th ventricle; cc, central canal; Com, commissural subnucleus of the NTS; DMV, dorsal motor nucleus of the vagus; cc, central canal; Slt, dorsolateral subnucleus of the NTS; Sm, medial subnucleus of the NTS; St, tractus solitarius; GR, nucleus gracilis; Cu, cuneate nucleus; Ecu, external cuneate nucleus. Calibration mark, 500 mm.
?
hcrt-1 into a site in the dorsolateral NTS in an OVX1E rat (Fig. 5a) and at a corresponding site in the OVX only animal (Fig. 5b), the durations of both the depressor and bradycardia responses elicited in the OVX1E animals (OVX1E, MAP duration 211.1644.6 s, HR duration 472.0645.1 s; OVX only, MAP duration 144.3628.5 s, HR duration 192.5651.2 s) were significantly longer. In the OVX only animals, microinjections of the higher doses of hcrt-1 (2.5 pmol) into a few sites in the caudal medial and commissural subnuclei of the NTS complex
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Fig. 4. Bar chart showing the effect of microinjection of varying amounts of hcrt-1 (0.5–10 pmol) into the dorsolateral and medial subnuclei of the NTS on the magnitude of the mean arterial pressure (MAP; top panel) and heart rate (HR; bottom panel) response in OVX1E (hatched bars) and OVX only (open bars) female rats. All values are means6S.E. of the mean. Numbers in parentheses indicate number of NTS sites injected at that specific dose of hcrt-1. Note that the magnitude of the bradycardia response at all dosages was significantly greater (*P,0.05) in OVX1E animals than in OVX animals.
were found to elicit increases in MAP (n57; 11.462.6 mmHg) and / or HR (n52; 15.065.0 bpm) (Fig. 3). Similar responses were occasionally observed with injections of a 10 pmol dose of hcrt-1 into the commissural nucleus. Injection of hcrt-1 into areas immediately outside the NTS complex in both the OVX1E and the OVX only rats did not elicit any cardiovascular response (Fig. 3). In addition, injections of similar volumes of saline, the vehicle, into these NTS sites did not elicit cardiovascular responses. The effect of hcrt-1 microinjection into the dorsolateral or medial subnuclei of the NTS on the reflex bradycardia to activation of arterial baroreceptors following an acute rise in systemic AP was investigated in the OVX only and the OVX1E anaesthetized female rat (Fig. 6). Activation of NTS neurons by hcrt-1 significantly potentiated the reflex decrease in HR to baroreceptor activation in the OVX1E animals (Fig. 6, top panel). On the other hand, no effect was observed on the reflex bradycardia in the OVX only rats (Fig. 6, bottom panel).
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Fig. 5. Representative heart rate (HR), arterial pressure (AP), and mean AP (MAP) responses to a microinjection of 2.5 pmol of hcrt-1 (arrows) into the caudal dorsolateral NTS in OVX1E (a) and OVX only (b) rats. Note that the HR response is both of greater amplitude and duration in the OVX1E animal compared to the OVX only rat. On the other hand, although the magnitude of the depressor response in both the OVX1E and OVX only animals appears to be the same, the duration of the response is longer in the OVX1E animal. Calibration mark, 1 min.
4. Discussion Hcrt-containing neurons, located exclusively within the lateral hypothalamus [13,30,47,50], have been shown to extensively innervate different levels of the neuroaxis [18,30,35,47,48], including the NTS complex in the male rat [10,18,30]. The NTS is an area well known to be involved in reflex regulation of cardiac rate and vasomotor tone [7]. This study has demonstrated that injections of hcrt-1 into the NTS of the female rat elicited dose-related bradycardia and depressor responses that were greater in magnitude and / or duration in the OVX1E group compared with the OVX only animals. In addition, the data show that hcrt-1 in the NTS potentiated the reflex bradycardia to activation of the arterial baroreflex in the OVX1E rats only, suggesting that the neuronal circuits involved in these cardiovascular responses to activation of hcrt-1 are modulated by the circulating levels of E. The distribution and density of hcrt-1 immunoreactivity within the NTS complex was not altered by the circulating level of E in the OVX female. In both OVX only and in OVX1E-treated female rats, hcrt-1-labelled axons and axon terminals were found to innervate similar subnuclei of the NTS complex. However, it is interesting to note that by comparing Fig. 1 in the report by de Oliveira et al. [11] with Fig. 1 presented in this study on the distribution of hcrt-1 immunoreactivity within the NTS complex, it is readily apparent that, in the male, both the distribution and density of hcrt-1 labelling is different compared to the females. In the female, all structures in the NTS complex appear to receive a less dense innervation by hcrt-1 axons. In addition, the dorsal motor nucleus of the vagus was
Fig. 6. Graph showing the effect of hcrt-1 microinjection into the dorsolateral subnucleus of the nucleus of the solitary tract complex on the reflex bradycardia elicited by activation of arterial baroreceptors following an acute increase in mean arterial pressure (MAP) resulting from an i.v. injection of phenylephrine in OVX1E animals (top graph) and OVX only animals (bottom graph). Note that the HR component of the baroreceptor reflex is significantly (P,0.05) increased only in the OVX1 E rats at about 0.5 min (P,0.05) after the hcrt-1 injection. The reflex HR response begins to recover towards control values by 2.5 min. n, number of responses used to calculate each point plotted on the graph. All values are means6S.E.M. Control (d; 5 min before hcrt-1 injection; OVX1E, R 2 5 0.8194; OVX only, R 2 5 0.9945). Time after hcrt-1 injections: 0.5 min (m; OVX1E, R 2 5 0.8888; OVX, R 2 5 0.8707), and 2.5 min (j; OVX1E, R 2 5 0.9966; OVX only, R 2 5 0.0.7937).
innervated by hcrt-1-labelled axons in the male, while hcrt-1 labelling within this structure in the female was not apparent. In support of this latter observation, recently, in the male rat, hcrt-1 has been shown to evoke gastric acid secretion [45] and to directly excite the dorsal motor nucleus of the vagus motor neurons in vitro [21]. The dorsal motor nucleus of the vagus is known to be involved in the parasympathetic regulation of gastrointestinal function [23]. These data suggest not only a gender difference
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in the innervation of the dorsal vagal complex, but also in the function of hcrt-1 in this area between the female and male. The finding that activation of the dorsolateral and medial subnuclei of the NTS complex, regions that have previously been shown to receive baroreceptor afferent projections [7], elicits bradycardia and depressor responses and alters the baroreflex was not unexpected, as these cardiovascular responses elicited by hcrt-1 into the NTS are consistent with those observed during electrical or glutamate stimulation of this NTS region, and during activation of the baroreceptor reflex [7,26]. In addition, the responses to activation of the NTS neurons by hcrt-1 in the female rat are qualitatively similar to those reported in an earlier study in the male rat in which hcrt-1 injection into the dorsolateral and medial subnuclei of the NTS complex elicited bradycardia and depressor responses [11]. However, it is interesting to note in this study that circulating E within physiological levels (approximately 30 pg / ml plasma) affected the magnitude of these cardiovascular responses. The HR and MAP responses were greater in magnitude in OVX1E animals compared to OVX only animals. In addition, the responses in the OVX1E animals appear to be smaller in magnitude than those previously reported in the male rat [11]. Although a direct comparison between the magnitude of the cardiovascular responses in the two different sexes was not made, it is evident that the responses elicited in the male [11] were generally about 30% greater than in the OVX1E female. This finding is consistent with our observation that the density of hcrt-1 immunoreactivity in the NTS complex of either the OVX only or OVX1E female is considerably less than that in the male [11]. Taken together, these data suggest not only that a gender difference exists between the functional innervation of the NTS by hcrt-1 pathways, but also that the levels of plasma E influence the sensitivity of neurons involved in the reflex control of the circulation to hcrt-1. In addition, these data suggest that the differences observed in this study between OVX only and OVX1E females in the magnitude of the cardiovascular responses to hcrt-1 is likely not due to the innervation of the NTS by hcrt-1 axons, but may reflect changes in the expression of hcrt-1 receptors on NTS neurons in the OVX1E female rat. Although the mechanism by which E may alter the neuronal circuits in the NTS to potentiate the bradycardia response to hcrt-1 in the OVX1E animals is not known, it has been demonstrated that neurons within the caudal NTS complex contain E receptors [25], and injections of E into the NTS complex produce a decrease in resting arterial pressure and potentiate the reflex bradycardia to activation of arterial baroreceptors [36–38]. In addition, it has been reported that E treatment in the OVX mouse can facilitate the baroreceptor reflex [32]. Furthermore, it has been shown that E can alter the release of glutamate [28], a finding that has also been suggested as a mechanism of action for hcrt-1 in the hypothalamus [44] and in NTS
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neurons [43]. These data would therefore suggest that hcrt-1 may act to either presynaptically increase the release of glutamate [43,44], the putative neurotransmitter in baroreceptor afferent fibers [31], or to interact with E sensitive interneurons in the NTS complex that facilitate the baroreflex. It has been reported that the bradycardia response elicited by hcrt-1 microinjection into the NTS in the male rat is mediated predominantly by the activation of central vagal cardiomotor neurons [11]. E has been shown to facilitate parasympathetic activity to the heart by potentiating the effect of baroreceptor afferent inputs into the nucleus ambiguus [36–38]. Therefore, the finding in this study that the HR response to activation of the baroreflex, which is mainly due to vagal activation [9], during hcrt-1 injections was potentiated only in the E-treated animals suggests that hcrt-1 acts on central NTS circuits that are also modified by E. It was also observed that, in the OVX only animal, a small number of sites located in the commissural subnucleus of the NTS elicited tachycardia and pressor responses, an observation supported by an earlier study that identified pressor sites to hcrt-1 in the commissural nucleus of NTS [11,40]. The caudal commissural nucleus of the NTS complex has been shown to receive mainly chemoreceptor afferent inputs [7]. Taken together, these data suggest that hcrt-1 may be involved in the activation of neuronal systems in the NTS region that may mediate sympathoexcitatory responses to chemoreceptor activation. However, it should be noted that pressor sites to hcrt-1 injections were not found within the NTS complex of OVX1E animals. Therefore, these data suggest that the circulating level of E may act to inhibit neuronal systems in the NTS activated by hcrt-1 that increase sympathetic nervous system activity. In summary, data have been presented indicating that microinjections of hcrt-1 into the NTS elicit bradycardia and arterial pressure depressor responses that are greater in the OVX female treated with E compared to the non-Etreated OVX female. In addition, it has been shown that hcrt-1 potentiates the baroreceptor reflex in these E-treated females. Taken together, the data suggest that E alters the sensitivity of NTS mechanisms involved in the reflex regulation of the circulation to hcrt-1.
Acknowledgements This work was supported by a research grant from the Heart and Stroke Foundation of Ontario to Dr. J. Ciriello.
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