Cardiovascular responses to microinjection of nociceptin and endomorphin-1 into the nucleus tractus solitarii in conscious rats

Neuroscience 132 (2005) 1009 –1015

CARDIOVASCULAR RESPONSES TO MICROINJECTION OF NOCICEPTIN AND ENDOMORPHIN-1 INTO THE NUCLEUS TRACTUS SOLITARII IN CONSCIOUS RATS L. MAOa* AND J. Q. WANGa,b,c

example, systemic injection of nociceptin or its analog [Tyr1]nociceptin decreased blood pressure in anesthetized rats (Champion and Kadowitz, 1997a,b). The decrease in blood pressure might be mediated by the selective stimulation of the nociceptin receptor, i.e. the opioid receptor-like receptor, given the fact that nociceptin-induced responses were resistant to blockade of the traditional opioid receptors with the nonselective antagonist naloxone (Champion and Kadowitz, 1997b). In addition to the depressor effect observed in anesthetized state, the same cardiovascular effect after systemic administration of nociceptin was also observed in conscious mice (Madeddu et al., 1999). In addition to peripheral effects, nociceptin may act centrally to modulate cardiovascular activities. An immunohistochemistry study showed that a moderate to high density of nociceptin receptor staining was present throughout the brainstem (Anton et al., 1996). Furthermore, nociceptin peptides and mRNAs have been identified in the medullary regions that participate in cardiovascular regulation (Neal et al., 1999). The previous studies in this laboratory have shown that 1) nociceptin inhibited spontaneous discharges of neurons recorded from the rostral ventrolateral medulla (RVL) in vitro (Chu et al., 1998, 1999a) and 2) local injection of nociceptin into the RVL consistently induced depressor and bradycardic responses (Chu et al., 1999b). Moreover, injection of nociceptin into the nucleus tractus solitarii (NTS), another important area in the medulla for control of cardiovascular activity and reflex, produced increases in blood pressure and heart rate (HR) in anesthetized rats (Mao and Wang, 2000). These results indicate that nociceptin is among neuropeptides in the RVL and NTS that significantly regulate peripheral cardiovascular activity. The ␮-opioid receptor has a widespread distribution throughout the CNS, including the NTS (Aicher et al., 2000), and is involved in the regulation of a broad range of functional activities (Mansour et al., 1995). Recently, a tetrapeptide, endomorphin-1 (EM-1; Tyr-Pro-Trp-Phe-NH2), has been identified from rat brain tissue (Zadina et al., 1997). This peptide has been reported to be the endogenous ligand for ␮-opioid receptors (Zadina et al., 1997). To examine putative roles of EM-1 in the central modulation of cardiovascular activity and reflex, we recently found that EM-1 profoundly inhibited electrical activity of cardiovascular neurons in the RVL in vitro (Chu et al., 1999a). Thus, EM-1-sensitive opioid ␮ receptors possess the ability to modulate RVL neurons. However, to date, no attempt has been made to define the role of EM-1 in the regulation of

a

Department of Basic Medical Science, University of Missouri–Kansas City, School of Medicine, Kansas City, MO 64108, USA b Department of Anesthesiology, Saint Luke’s Hospital of Kansas City, Kansas City, MO 64111, USA c

Department of Pharmacology, University of Missouri–Kansas City, School of Pharmacy, Kansas City, MO 64108, USA

Abstract—Increasing evidence suggests an active participation of nociceptinergic transmission in the central control of cardiovascular activity and reflex. In this study, the role of the classic opioid ␮ receptor and the nociceptin/orphanin FQ receptor, a novel opioid receptor, in the nucleus tractus solitarii (NTS) in the regulation of cardiovascular activity was investigated and compared in chronically cannulated and freely moving conscious rats. Microinjections of nociceptin, an endogenous ligand for the nociceptin receptor, into the relatively rostral NTS produced dose-related (0.04, 0.2, and 1 nmol) increases in blood pressure and heart rate. Intra-NTS injection of the selective nociceptin receptor antagonist [Nphe1]Nociceptin(1–13)NH2 (NOR-AN) at 1 nmol blocked the increases in blood pressure and heart rate induced by nociceptin. In contrast, pretreatment with the nonselective opioid receptor antagonist naloxone (5 nmol) had no effects on the cardiovascular responses to nociceptin. Like nociceptin, microinjection of endomorphin-1 (EM-1), an endogenous ligand for the opioid ␮ receptor, into the rostral NTS increased blood pressure and heart rate in a dose-dependent manner (0.04, 0.2, and 1 nmol). Pretreatment with naloxone (5 nmol), but not NOR-AN, blocked cardiovascular responses elicited by EM-1. Neither NOR-AN nor naloxone alone had significant effects on the baseline blood pressure and heart rate. Injection of excitatory amino acid L-glutamate (1 nmol) into the same sites caused the typical depressor and bradycardic responses. In the caudal NTS areas, nociceptin and EM-1 seemed to induce opposite responses: hypotension and bradycardia. These results suggest that the novel nociceptin receptors and traditional opioid receptors in the NTS may be independently involved in the regulation of cardiovascular activity. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: arterial pressure, heart rate, glutamate, opioid receptor, orphanin FQ, naloxone.

Recent studies suggest that nociceptin exerts a strong influence over cardiovascular activity and reflexes. For *Corresponding author. Tel: ⫹1-816-235-1795; fax: ⫹1-816-235-1776. E-mail address: [email protected] (L. Mao). Abbreviations: ABP, arterial blood pressure; ACSF, artificial cerebrospinal fluid; bpm, beats per minute; EM-1, endomorphin-1; HR, heart rate; MAP, mean arterial pressure; NOR-AN, [Nphe1]nociceptin(1–13) NH2; NTS, nucleus tractus solitarii; RVL, rostral ventrolateral medulla. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.01.037

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activity of NTS neurons in vivo, despite the existence of the peptide in the NTS (Martin-Schild et al., 1999). The present study was therefore designed to evaluate the role of EM-1-sensitive ␮ receptors in the NTS in the modulation of cardiovascular activity. In particular, the peripheral cardiovascular responses to EM-1 injected into the NTS were compared with those induced by the synthetic peptide agonist nociceptin. Pharmacological blockade of nociceptin or ␮-opioid receptors was achieved by microinjection of the antagonist [Nphe1]nociceptin(1–13)NH2 (NOR-AN; Berger et al., 2000; Calo et al., 2000) or naloxone, respectively. All studies were performed in chronically cannulated and conscious rats in order to prevent a possible interference of anesthetics.

EXPERIMENTAL PROCEDURES Animals Adult male Sprague–Dawley rats (250 –300 g; Charles River, New York, NY, USA) were housed in a plastic cage in a colony room maintained on a 12-h light/dark cycle in a controlled environment at a constant temperature of 23 °C and humidity of 50⫾10% with food and water provided ad libitum. Animals were kept in our animal facility for at least 5 days before the experiment. All animal use procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. All efforts were made to minimize both the suffering and the number of animals used.

Surgical procedures Chronic implantation of a guide cannula that allowed microinjections to be made into one side of the NTS of conscious, freely moving rats was performed as described previously (Mao and Abdel-Rahman, 1996; Mao and Wang, 2000). Briefly, rats were anesthetized with 4% chloral hydrate (400 mg/kg, i.p.) and placed in a David Kopf stereotaxic frame in a prone position. A 23-gauge stainless steel guide cannula (20 mm in length) was implanted from the dorsal surface of the cranium according to the following coordinates: 13.3 mm caudal to bregma, 0.6 mm lateral to midline, and 6.0 mm dorsal to surface of skull. The guide cannula was permanently secured to the skull by two jeweler’s screws and dental cement. An inner stainless steel wire of the same length was then lowered through the guide cannula, effectively sealing the channel until the day of the experiment. Two to three days after the initial surgery, the animals were reanesthetized with 4% chloral hydrate (400 mg/kg, i.p.). PE-50 catheters, filled with heparinized saline (100 U/ml), were placed in the abdominal aorta and inferior vena cave via left or right femoral artery and vein for measurement of blood pressure and i.v. injection, respectively. The catheters were tunneled s.c., exteriorized at the back of the neck, flushed with a solution of heparin (100 U/ml), and sealed with stainless steel pins. The wound was closed by surgical clips and swabbed with povidone-iodine solution. The experiment was performed at least 3– 4 days after the second surgery, i.e. 5–7 days after brain surgery.

Measurement of blood pressure and HR On the day of the experiment, animals were brought to a quiet room for 2–3 h before experiment. The catheter for arterial blood pressure (ABP) measurement was connected to a CE 344 pressure transducer (Maxxim Medical, Athens, TX, USA). Mean arterial pressure (MAP) and HR were derived electronically from the ABP pulses. The three parameters (ABP, MAP, and HR) were

monitored and recorded through a “real-time” ADInstruments PowerLab/8s data recording and analysis system on a PowerMac computer. Blood pressure and HR were allowed to stabilize for at least 30 min prior to the experiment.

Intra-NTS microinjections Microinjections were made directly into the NTS in conscious freely moving rats through a 30-gauge stainless steel injector. This injector replaced the inner steel wire and extended 2 mm beyond the previous implanted guide cannula. The injections of drugs were made at a volume of 80 nl over 15 s. Before or, in a few cases, after each of the experiments, L-glutamate (1 nmol/80 nl) was injected into the NTS site. The injection sites were functionally considered to be localized within the cardiovascular zone of the NTS if the typical depressor and bradycardic responses were induced after L-glutamate injection (Talman et al., 1980; Leone and Gordon, 1989). Pontamine Sky Blue in a volume of 80 nl was injected in the same sites for histological verification of injection sites. Animals were killed with a lethal dose of the anesthetic given intraperitoneally and were decapitated. The brain was removed and fixed in a solution of 10% formalin for 4 –7 days. Frozen serial frontal sections (40 ␮m) of the brainstem were cut, mounted, and stained with Neutral Red, from which the actual injection sites were identified by referring to the Paxinos and Watson (1986) atlas.

Drugs Chloral hydrate and L-glutamate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). EM-1, nociceptin, NOR-AN, and naloxone hydrochloride were purchased from Tocris Cookson (Ballwin, MO, USA). Chloral hydrate was dissolved in 0.9% sodium chloride. L-Glutamate, EM-1, nociceptin, NOR-AN, and naloxone were dissolved in artificial cerebrospinal fluid (ACSF; in mM: NaCl 123, CaCl2 0.86, KCl 3.0, MgCl2 0.89, NaHCO3 25, NaH2PO4 0.50, and Na2HPO4 0.25, aerated with 95% O2–5% CO2, pH 7.4). All drugs were freshly prepared immediately before use.

Statistical analysis The results are presented as means⫾S.E.M. Data were analyzed using a nested two-way analysis of variance (ANOVA) followed by a group comparison with least square-adjusted means. The criterion for statistical significance was P⬍0.05.

RESULTS Effects of nociceptin or EM-1 on baseline blood pressure and HR Baseline levels of MAP and HR of conscious, freely moving rats were 107.4⫾2.1 mm Hg and 382.8⫾11.2 beats per minute (bpm; n⫽35). In time control experiments, without drug injection, continuous measurements of MAP and HR were made in conscious rats over 1 h, a duration that all experiments in this study conformed to. The data collected during this 1 h indicated only minimal fluctuations of the two parameters (data not shown). Injection of nociceptin into the rostral NTS caused dose-dependent increases in MAP and HR (Fig. 1). In a dose range from 0.04 to 1 nmol, a lower dose of nociceptin (0.04 nmol) did not produce any detectable changes. At the middle dose (0.2 nmol), nociceptin started to increase MAP and HR. Greater responses were induced after nociceptin was injected at a higher dose (1 nmol). Significant elevation of MAP and HR was

L. Mao and J. Q. Wang / Neuroscience 132 (2005) 1009 –1015

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Effects of intra-NTS injection of NOR-AN or naloxone on baseline blood pressure and HR Effects of pharmacological blockade of nociceptin or ␮opioid receptors with the selective nociceptin antagonist NOR-AN or the non-selective opioid receptor antagonist naloxone, respectively, on basal levels of blood pressure and HR were tested in five groups of rats (n⫽4 per group). A unilateral injection of 0.2 nmol of NOR-AN into the rostral NTS produced no significant alteration in blood pressure and HR during a 30-min observation compared with the group of rats treated with ACSF (data not shown). At a higher dose (1 nmol), NOR-AN still did not have any detectable effects on either blood pressure or HR (data not shown). Similarly, intra-NTS injection of naloxone at a lower (1 nmol) or higher (5 nmol) dose had no effect on baseline blood pressure and HR (data not shown).

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Time after injection (min) Fig. 1. Changes in MAP (A) and HR (B) after a unilateral injection of ACSF or nociceptin (0.04, 0.2, or 1 nmol/80 nl) into the NTS in conscious rats (n⫽4 –11). Note that nociceptin induced dose- and time-dependent increases in MAP and HR. Values are expressed as mean changes from baseline MAP and HR and vertical bars indicate SEMs. * P⬍0.05 compared with the corresponding values taken from ACSF group.

seen immediately, peaked at 2 min, and returned to preinjection levels approximately 5–10 min after injection. Similarly, injection of EM-1 at the some dose range (0.04, 0.2, and 1 nmol) produced dose-dependent increases in MAP and HR (Fig. 2). Microinjection of EM-1 at a higher dose (1 nmol) produced long and stable increases in MAP and HR, which lasted approximately for 5–10 min. In some animals, repeat injections of nociceptin or EM-1 at 1 nmol were made into the same sites (two to three injections per site with 1 h interval); comparable responses of blood pressure and HR were observed, indicating the lack of rapid tachyphylaxis. To examine whether the cardiovascular responses to intra-NTS injection of nociceptin or EM-1 were due to leakage of the drug from the central injection site to peripheral circulation, i.v. injections of nociceptin (1 nmol/0.1 ml) and EM-1 (1 nmol/0.1 ml) were made in two separate groups of rats (n⫽5 per group). No changes in baseline MAP and HR were seen after systemic administration of nociceptin and EM-1 at the dose surveyed (data not shown). Fig. 3 illustrates actual traces of one example experiment showing the character of cardiovascular responses to

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Time after injection (min) Fig. 2. Changes in MAP (A) and HR (B) after a unilateral injection of ACSF or EM-1 (0.04, 0.2, or 1 nmol/80 nl) into the NTS in conscious rats (n⫽4 –10). Note that EM-1 induced dose- and time-dependent increases in MAP and HR. Values are expressed as mean changes from baseline MAP and HR and vertical bars indicate SEMs. * P⬍0.05 compared with the corresponding values taken from ACSF group.

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2 min Fig. 3. Representative recordings showing alterations in hemodynamic activity in response to microinjection of ACSF, nociceptin (1 nmol), EM-1 (1 nmol), or L-glutamate (1 nmol) into the same site of the NTS in a conscious rat. The tracings from top to bottom are pulsatile ABP, MAP, and HR, respectively. Arrow indicates the start of the drug injection. A transient pressor response was induced following a single injection of nociceptin or EM-1 at 1 nmol whereas a transient depressor response was induced following an injection of L-glutamate into the same site at 1 nmol.

Effects of NOR-AN or naloxone on the cardiovascular responses to intra-NTS injection of nociceptin To verify the specificity of nociceptin receptors in mediating nociceptin action, the effects of intra-NTS injection of ACSF, NOR-AN, or naloxone on the cardiovascular responses to nociceptin were investigated in three different groups of rats. Each rat served as its own control and received a single dose of nociceptin (1 nmol) before and 10 min after ACSF, NOR-AN (1 nmol), or naloxone (5 nmol). ACSF did not influence the cardiovascular responses to nociceptin (Fig. 4). However, 10 min following intra-NTS injection of NOR-AN (1 nmol), the pressor responses to nociceptin were totally blocked (Fig. 4). Similarly, the tachycardic responses were blocked by NOR-AN (Fig. 4). The pressor and tachycardic responses to nociceptin resumed 1 h after the termination of NOR-AN injection (data not shown), indicating recovery of cardiovascular responses after blockade. In contrast to NOR-AN, naloxone (5 nmol) had no effects on cardiovascular responses to nociceptin (Fig. 4). Effects of NOR-AN or naloxone on the cardiovascular responses to intra-NTS injection of EM-1 In a separate study, the effects of intra-NTS injection of ACSF, NOR-AN, and naloxone on the cardiovascular responses to EM-1 were investigated. As shown in Fig. 5, intra-NTS injection of EM-1 (1 nmol) produced pressor and tachycardic responses. These responses were not influenced by ACSF microinjected into the NTS. However, the

pressor responses elicited by EM-1 were significantly attenuated 10 min after injection of naloxone (5 nmol), but not NOR-AN (1 nmol). The pressor responses to EM-1 were recovered 1 h after the naloxone injection (data not shown). The tachycardic responses to EM-1 were not significantly changed after naloxone, NOR-AN, or ACSF microinjection (Fig. 5). From the histological examination, the injection sites from the aforementioned studies were distributed in the ventral and medial parts of relatively rostral NTS (Fig. 6A), in which injection of L-glutamate (1 nmol) caused typical depressor and bradycardic responses whereas injection of nociceptin or EM-1 caused pressor and tachycardic responses. In addition, cardiovascular responses to nociceptin or EM-1 were tested in relatively caudal NTS sites of five rats. In almost all sites tested in the caudal NTS (seven of eight sites in five rats, Fig. 6B), injection of nociceptin (1 nmol, 2 min) decreased MAP by ⫺16.8⫾3.2 (P⬍0.05 vs. preinjection value) mm Hg and HR by ⫺29.7⫾6.1 (P⬍0.05 vs. preinjection value) bpm. Similar results were obtained following injections of EM-1 (1 nmol, 5 min) into the most of those sites (data not shown).

DISCUSSION The present study investigated the roles of the newly discovered nociceptinergic system and the classic opioid system in the NTS in the regulation of peripheral cardiovascular activity in conscious rats. The main findings of this investigation are: 1) pharmacological activation of nociceptin or ␮-opioid receptors in the rostral NTS with a direct injection of synthetic nociceptin or EM-1 respectively sig-

L. Mao and J. Q. Wang / Neuroscience 132 (2005) 1009 –1015

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Fig. 4. Effects of the nociceptin receptor antagonist NOR-AN and the opioid receptor antagonist naloxone on the nociceptin-induced cardiovascular responses. Changes in MAP (A) and HR (B) following injection of nociceptin (1 nmol) into the NTS were measured in conscious rats before and 10 min after intra-NTS injection of NOR-AN (1 nmol, n⫽6), naloxone (5 nmol, n⫽6), or equal volume of ACSF (n⫽6). Note that nociceptin-induced increases in MAP and HR were blocked only by NOR-AN. Values are expressed as mean changes from baseline MAP and HR and vertical bars indicate SEMs. * P⬍0.05 compared with the corresponding control (pretreatment) values.

nificantly increased blood pressure and HR; 2) the nociceptin-induced increases in blood pressure and HR were blocked by the selective nociceptin receptor antagonist NOR-AN, but not by non-selective opioid antagonist naloxone; 3) the EM-1-induced increases in blood pressure and HR were blocked by naloxone, but not by NOR-AN. These findings provide the direct evidence that nociceptin and ␮-opioid receptors in the rostral NTS inhibit NTS neurons to regulate cardiovascular activity. Since results were obtained in conscious and unrestrained rats, inhibitory effects of anesthetics on central cardiovascular neurons (Fluckiger et al., 1985; Bedran-de-Castro et al., 1990) and potential interfering interactions between anesthetics and nociceptin or EM-1 have been circumvented. Finally, pharmacological blockade of nociceptin or ␮-opioid receptors had no effects on baseline blood pressure and HR. This suggests that, in the NTS, nociceptin and ␮-opioid receptors may not be tonically active for maintaining normal cardiovascular activity.

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The NTS areas that participate in cardiovascular regulation include the baroreceptor projection site located in the midline area of the commissural subnucleus of NTS and the intermediate portion of NTS (Leone and Gordon, 1989). In this study, microinjections of nociceptin and EM-1 were made into an area relatively rostral to the baroreceptor afferent zone in the NTS. These injections elicited marked pressor and tachycardic responses. These data are consistent with those observed in a large number of previous reports in which injection of opioid agents into this area induced significant changes in blood pressure and HR in anesthetized rodents (Gordon, 1990; Rabkin, 1993; Mao and Wang, 2000; Sapru and Chitravanshi, 2002). In addition, the cardiovascular effect of nociceptin and EM-1 was found to be regionspecific. While nociceptin, EM-1, or EM-2 injected into the relatively rostral NTS area caused pressor and tachycardic responses (Sapru and Chitravanshi, 2002; this study), injection of those ligands into the relatively caudal NTS caused depressor and bradycardic responses (Kasamatsu et al., 2004; this study).

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0 Fig. 5. Effects of the nociceptin receptor antagonist NOR-AN and the opioid receptor antagonist naloxone on the EM-1-induced cardiovascular responses. Changes in MAP (A) and HR (B) following injection of EM-1 (1 nmol) into the NTS were measured in conscious rats before and 10 min after intra-NTS injection of NOR-AN (1 nmol, n⫽5), naloxone (5 nmol, n⫽6), or equal volume of ACSF (n⫽5). Note that EM-1-induced increases in MAP were blocked only by naloxone. Values are expressed as mean changes from baseline MAP and HR and vertical bars indicate SEMs. * P⬍0.05 compared with the corresponding control (pretreatment) values.

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1 mm Fig. 6. Schematic diagrams of coronal sections of rat brain stem showing the microinjection sites (solid circles) in the NTS. (A) The microinjection sites where injection of nociceptin and EM-1 caused pressor and tachycardic responses. (B) The microinjection sites where injection of nociceptin and EM-1 caused depressor and bradycardic responses. Cu, cuneate nucleus; Sp5C, caudal part of spinal trigeminal nucleus; Sp5I, interpolar part of spinal trigeminal nucleus.

Different mechanisms may underlie pressor and depressor responses caused by injection of nociceptin and EM peptides into the different sites of NTS. In the rostral NTS, direct inhibition of NTS neurons may be largely responsible for pressor and tachycardic responses to nociceptin and EM injection. In the caudal NTS, an indirect mechanism may be involved in depressor and bradycardic responses to nociceptin and EM peptides. Kasamatsu et al. (2004) observed that the antagonist selective for ionotropic glutamate receptors (both NMDA and nonNMDA subtypes) or GABA receptor blocked depressor and bradycardic responses to microinjection of EM-2 into the NTS. Thus, EM-2 may suppress the local inhibitory GABAergic transmission, which results in disinhibition of glutamatergic transmission at ionotropic receptors, causing depressor and bradycardic responses (Kasamatsu et al., 2004). In addition, previous studies found that both nociceptin and endomorphin inhibited electrical activity of rat RVL neurons in vitro, indicating RVL is another medul-

lary area to mediate cardiovascular effects of nociceptin and endomorphin (Chu et al., 1998, 1999c). Nociceptin is considered to be a novel member of the opioid peptide family based on its homology in amino acid sequence and similarity of its functional roles in the regulation of a variety of physiological activities, especially pain and cardiovascular modulation, with the typical opioid peptides. For cardiovascular regulation, the majority of responses to nociceptin receptor stimulation in the rostral NTS are increases in blood pressure and HR. Although these responses are similar to the effects of opioid peptide in the NTS on cardiovascular activity, nociceptin effects were antagonized by NOR-AN but not naloxone, and opioid peptide effects were antagonized by naloxone but not NOR-AN. This suggests that nociceptin and traditional opioid peptides are two independent systems in the NTS for controlling cardiovascular functions. The cardiovascular responses induced by nociceptin are similar to the previous data obtained from anesthetized rats by this laboratory and others (Mao and Wang, 2000; Sapru and Chitravanshi, 2002). However, the changes in blood pressure and HR in conscious rats were seen more early, and returned to the preinjection level much faster than in anesthetized rats, suggesting anesthetics may have a powerful influence on the nociceptin-induced cardiovascular responses. Perhaps the inhibitory influence of anesthetics on baroreflex sensitivity (Fluckiger et al., 1985; Bedran-de-Castro et al., 1990) may delay recovery of changes induced by nociceptin. While the conscious animal model used in this study has its obvious advantages, it also has its disadvantages. For example, the drugs injected into the NTS may elicit respiratory responses, and observed cardiovascular responses might be secondary to respiratory changes. Since we did not simultaneously monitor respiratory responses, it is unknown whether the drugs injected into the NTS alter respiratory activity and whether altered activity, if there is any, contributes to the cardiovascular changes. In conclusion, the present study found that a unilateral injection of nociceptin or EM-1 into the rostral NTS elevated blood pressure and HR. The elevation induced by nociceptin was sensitive to the nociceptin receptor antagonist but not to the opioid receptor antagonist. Similarly, the elevation induced by EM-1 was sensitive to the opioid receptor antagonist but not to the nociceptin receptor antagonist. Intra-NTS injection of the nociceptin receptor antagonist or the opioid receptor antagonist alone did not affect the baseline blood pressure and HR, a similar result observed in anesthetized state (Gordon, 1990; Shah et al., 2003). In the caudal NTS areas, nociceptin and EM-1 seemed to induce opposite responses: hypotension and bradycardia. These data obtained in conscious rats demonstrate that the nociceptin and opioid receptors in the NTS are not tonically active in the regulation of the basal operation of cardiovascular activity under normal physiological conditions. However, upon activation, the two receptors showed a strong influence over peripheral cardiovascular activity.

L. Mao and J. Q. Wang / Neuroscience 132 (2005) 1009 –1015 Acknowledgments—This work was supported by NIH grants (DA10355 and MH61469) and a grant 0265239Z from the American Heart Association.

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(Accepted 25 January 2005) (Available online 24 March 2005)