Brain Research 984 (2003) 93–103www.elsevier.com/ locate/brainres

Research report

C ardiovascular responses to microinjections of nociceptin into amidline area in the commissural subnucleus of the nucleus tractus

solitarius of the rat*Neel Shah, Vineet C. Chitravanshi, Hreday N. Sapru

Department of Neurological Surgery, MSB H-586, New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103,USA

Accepted 4 June 2003

Abstract

Immunoreactivity for nociceptin, an endogenous ligand for the ORL1 opioid receptors, has been reported in the nucleus tractussolitarius (nTS). A midline area in the commissural subnucleus (nCom) of nTS is the site of peripheral chemoreceptor projections. Thisinvestigation was carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats, to study the cardiovascular effects ofthe activation of ORL1 receptors in a midline area of the nCom. Microinjections (30 nl) of nociceptin (0.15–0.62 mM) into the nCom

1elicited depressor and bradycardic responses. Prior microinjections of [N-Phe ]–nociceptin-(1–13)–NH (4.5 mM), a specific antagonist2

for ORL1 opioid receptors, into the nCom blocked the effects of nociceptin (0.31 mM, the maximally effective concentration), but notendomorphin-2 (0.6 mM; an endogenous ligand form-opioid receptors). On of other hand, naloxone (0.125 mM; an antagonist forclassical opioid receptors) did not block the effects of nociceptin, while it did block the effects of endomorphin-2. The blockade of

1nociceptin effects by [N-Phe ]–nociceptin-(1–13)–NH and endomorphin-2 by naloxone, was not due to some nonspecific effects2

because the responses toL-Glu (5 mM) remained unaltered after the microinjection of these antagonists. These results indicate thatactivation of ORL1 receptors in the nCom may play a role in the regulation of cardiovascular function. 2003 Elsevier B.V. All rights reserved.

Theme: Endocrine and autonomic regulation

Topic: Cardiovascular regulation

Keywords: Blood pressure; Bradycardia; Chemoreceptor projection site; Depressor response; Glutamate; Heart rate

1 . Introduction capeptide, showing a homology in amino acid sequencewith existing endogenous peptides especially dynorphin A,

Opioid receptors are G-protein coupled and have been is considered to be an endogenous ligand of ORL1classified into three major subtypes namelym (mu), d receptor because of its high and selective affinity for this(delta) andk (kappa) receptors[55,59]. The endogenous receptor and a very poor affinity form, d and k opioidligands for these subtypes of opioid receptors are endo- receptors[12,36]. This heptadecapeptide was named asmorphins[23,39,60],enkephalins and dynorphins[45,49], nociceptin[34,35] or Orphanin-FQ[46]. Naloxone blocksrespectively. A novel G-protein coupled receptor has been the effects ofm, d andk opioid receptor agonists[45,49]named opioid receptor like receptor (ORL1 receptor) but not those of ORL1 receptor agonist[7,8].because it shares a high sequence similarity withm, d and ORL1 is expressed throughout the brain and spinal cordk receptors [2,5,9,26,34,38]. Based on the proposed [1,5,20,37,38,41].The presence of immunoreactivity fornomenclature of opioid receptors, ORL1 receptor has also nociceptin has also been reported throughout the centralbeen named as opioid receptor OP[16,24]. A heptade- nervous system[17,40]. Examination of these reports4

indicates that ORL1 receptors and immunoreactivity fornociceptin are present in the nucleus tractus solitarius*Corresponding author. Tel.:11-973-972-5876; fax:11-973-972-(nTS), especially in the medial and commissural regions5986.

E-mail address: sapru@umdnj.edu(H.N. Sapru). [1,5,20,37,40,41].A midline area adjacent to the calamus

0006-8993/03/$ – see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0006-8993(03)03116-0

94 N. Shah et al. / Brain Research 984 (2003) 93–103

scriptorius (CS) in the commissural subnucleus of nTS monitored continuously and maintained at 3760.58C(nCom) has been named the chemoreceptor projection site using an infrared lamp connected to a temperature control-because of the presence of chemosensitive neurons and ler. All of the tracings were recorded on a polygraphprojections from carotid chemoreceptor afferents in this (Grass model 7D). In order to determine the role ofarea[10,11,19,56].Detailed and systematic studies on the parasympathetic innervation to the heart in mediating theeffects of ORL1 receptor activation by nociceptin in the HR responses elicited by microinjections of nociceptin intonCom have not been reported. However, preliminary the nCom, silk sutures were placed loosely around theresults of our studies on the effects of nociceptin in the vagus nerves bilaterally for subsequent identification andnCom were presented at a symposium held in Orlando, FL, sectioning of the nerves.USA. The proceedings of this symposium have beenpublished[48]. The present investigation was undertaken 2 .2. Microinjection techniqueto carry out a systematic study on the cardiovasculareffects of microinjections of nociceptin into the nCom. The rats were placed in a prone position in a stereotaxic

instrument (David Kopf Instruments, CA, USA) with thebite bar 18 mm below the interaural line. The medulla was

2 . Methods exposed by removing the dorsal neck muscles, incising theatlanto-occipital membrane and removing part of the

2 .1. General procedures occipital bone and dura. Multi-barreled glass micropipettes(tip size 20–40mm) were mounted on a micromanipulator

Experiments were done in adult male Wistar rats (David Kopf Instruments) and each barrel was connected(Charles River Labs., MA, USA) weighing 300–350 g. All via polyethylene tubing to one of the channels on aanimals were housed under controlled conditions with a 12 picospritzer (General Valve, Fairfield, NJ, USA). Theh light–dark cycle. Food and water were available to the contents of different barrels varied according to theanimals ad libitum. The experimental procedures were experiment being conducted. The coordinates for theperformed in accordance with NIH Guide for the Care and nCom, which is located in the midline, were: 0.3–0.5 mmUse of Laboratory Animals and the recommendations of caudal to the calamus scriptorius and 0.3–0.4 mm deepthe Panel on Euthanasia of the American Veterinary from the dorsal surface of the medulla. The micropipetteMedical Association. All protocols were approved by the was inserted into the nCom perpendicularly. The volumeInstitutional Animal Care and Use Committee at the of all microinjections (30 nl), as determined by theUniversity of Medicine and Dentistry of New Jersey, New displacement of fluid meniscus in the micropipette-barrel,Jersey Medical School, Newark, USA. was visually confirmed under a modified binocular

The animals were anesthetized by administering iso- horizontal microscope with a graduated reticule in oneflurane (3% in 100% oxygen) via a nose-mask. The trachea eye-piece (WPI, Florida, USA). The duration of eachwas cannulated with polyethylene tubing (PE 240) and microinjection was 10–20 s.anesthesia was maintained by tracheal administration ofisoflurane (2% in 100% oxygen). The rats were artificially 2 .3. Statistical analysesventilated using a rodent ventilator (Harvard Instruments,MA, USA, model 683). The femoral vein and artery on The means and standard error of the means (S.E.M.)one side were cannulated with polyethylene tubing (PE 50) were calculated for maximum changes in MAP and HR infor intravenous injections of various agents and for moni- response to microinjections of different concentrations oftoring blood pressure (BP), respectively. A pressure trans- nociceptin. Comparisons of MAP and HR responses toducer (Statham P23 Db) was used to measure the BP. The different concentrations of nociceptin were made by usingHR was monitored by a tachograph (Grass 7 P4) that was a one-way analysis of variance followed by Tukey–triggered by the BP waves. Urethane (1.2–1.4 g/kg) was Kramer multiple comparison test. Comparisons of theinjected intravenously in five aliquots at 3-min intervals. maximum changes in MAP and HR elicited by nociceptinThe tracheal inhalation of isoflurane was discontinued after orL-Glu before and after the administration of the ORL1the administration of the first aliquot of the urethane. The receptor antagonist were made by using pairedt-test. In alldepth of anesthesia was established by pinching the hind cases, the differences were considered significant atP,paw of the rat; absence of a BP response and/or withdraw- 0.05.al of the limb indicated that the rat was properly anes-thetized. The end-tidal CO was monitored by a continu- 2 .4. Drugs and chemicals2

ous measurement of expired gas with an infrared CO -2

analyzer modified for use in small animals (Micro-Cap- The following drugs were used:L-glutamate mono-nometer, Columbus Instruments, OH, USA) and main- sodium, endomorphin-2 (m-receptor agonist), isoflurane,

1tained between 3.5 and 4.5% by adjusting the frequency nociceptin (ORL1 receptor agonist), [N-Phe ]–nociceptin-and tidal volume on the ventilator. Rectal temperature was (1–13)–NH (ORL1 receptor antagonist), naloxone hydro-2

N. Shah et al. / Brain Research 984 (2003) 93–103 95

chloride (nonselective opioid receptor antagonist) and mM) were made at the same nCom site at 20-minurethane. All of the solutions for the microinjections were intervals. The decreases in MAP were 3065 and 3263.9freshly prepared in artificial cerebrospinal fluid (aCSF; pH mmHg for the first and fourth microinjection of nociceptin7.4). The composition of aCSF was as follows: NaCl (128 (0.31 mM), respectively (n55; P.0.05). Thus microinjec-mM), KCl (3 mM), CaCl (1.2 mM), MgCl (0.8 mM), tions of nociceptin repeated four times did not elicit2 2

dextrose (3.4 mM) and HEPES (5 mM). The control tachyphylaxis when the interval between microinjectionssolutions for the microinjections consisted aCSF. Where was at least 20 min.applicable, the concentration of drugs injected into the The role of vagus nerves in mediating the bradycardicnCom refers to their salts.L-Glutamate, endomorphin-2, responses elicited by microinjections of nociceptin in thenociceptin, naloxone and urethane were purchased from nCom was studied as follows. In these experiments (n54),

1Sigma-RBI (St. Louis, MO, USA). [N-Phe ]–nociceptin- silk sutures were placed loosely around the vagus nerves(1–13)–NH was purchased from Phoenix (Belmont, CA, bilaterally as described in Section 2.1. The nCom was2

USA). Isoflurane was purchased from Baxter (Deerfield, identified by a microinjection ofL-Glu (5 mM) (Fig. 1A).IL, USA). The chemicals for preparing aCSF were ob- Nociceptin (0.31 mM) was microinjected at the same site 5tained from Fisher (Atlanta, GA, USA). min later; the usual decreases in MAP (3265.1 mmHg)

and HR (2464 bpm) were elicited (typical response:Fig.1B). After a stabilization period of 20 min, the right vagus

3 . Results nerve was sectioned. Nociceptin (0.31 mM) was againmicroinjected 2 min later at the same site; there were no

The baseline values for MAP and HR in urethane- significant changes in BP and HR indicating that unilateralanesthetized rats used in this study were 110615 mmHg vagotomy did not alter the bradycardic responses toand 360630 bpm, respectively (n547). microinjections of nociceptin into the nCom (Fig. 1C).

After allowing a stabilization period of 20 min, the second3 .1. Concentration–response of nociceptin in the nCom vagus nerve was sectioned and within 2 min, nociceptin

(0.31 mM) was microinjected again at the same site;In this and other series of experiments, microinjections nociceptin-induced bradycardia was no longer present

of L-glutamate (L-Glu; 5 mM) were made to functionally indicating that bilateral vagotomy completely abolished theidentify the nCom from which increases in MAP bradycardic responses to nociceptin (Fig. 1D). Although(24.463.8 mmHg) and HR (29.464.9 bpm) were elicited the nociceptin-induced depressor responses persisted, there[10,11,56]. Nociceptin was microinjected at the same was a significant reduction in the decreases in BP; nocicep-nCom site through another barrel of the same micropipette. tin-induced decreases in MAP before and after bilateralA range of concentrations (0.15–0.62 mM) of nociceptin vagotomy were 3265.1 and 1862 mmHg, respectivelywas microinjected into the nCom. Only one microinjection (P,0.05).of nociceptin was made into the nCom of each animal withthe exception of series of experiments in which possible 3 .2. Effect of ORL1 receptor antagonist on nociceptintachyphylaxis to nociceptin microinjections or blockade of responsesnociceptin responses was studied. The decreases in MAP

1were 19.764, 32.363.9 and 20.763.7 mmHg in response [N-Phe ]–nociceptin-(1–13)–NH is a specific antago-2

to microinjections of 0.15, 0.31 and 0.62 mM concen- nist for ORL1 opioid receptors[6,22]. Microinjections oftrations of nociceptin, respectively (n59 for each con- L-Glu (5 mM) were used to identify the nCom from whichcentration). The decreases in HR were 23.263.2, 45.965.8 the pressor and tachycardic responses were elicited (Fig.and 2465.6 bpm in response to microinjections of 0.15, 2A). After an interval of 5 min, nociceptin (0.31 mM) was0.31 and 0.62 mM concentrations of nociceptin, respec- microinjected at the same site, and decreases in MAP andtively. Statistical calculations showed that the depressor as HR were observed which lasted for 3.1–5.7 min (Fig. 2B).well as bradycardic responses elicited by a 0.31 mM At 20 min after the BP and HR returned to control levels,concentration of nociceptin were significantly greater (P, the ORL1 receptor antagonist (4.5 mM) was microinjected0.05) than those elicited by 0.15 and 0.62 mM con- at the same site. In this and other series of experiments, thecentrations of nociceptin. Depending on the concentration microinjections of the ORL1 opioid receptor antagonistused, the onset and duration of nociceptin-induced car- alone into the nCom did not elicit any response (Fig. 2C).diovascular responses were 7.761.2 s and 5.760.98 min, After allowing a 2-min period for the ORL1 receptorrespectively. Since maximal depressor and bradycardic antagonist to diffuse so that it reached most of the nComresponses were elicited by the microinjection of 0.31 mM neurons, nociceptin (0.31 mM) was again injected at thenociceptin, this concentration was selected for further same site; the cardiovascular responses to nociceptin werestudy. completely blocked (Fig. 2D). The lack of responses to

In the studies on tachyphylaxis, four microinjections of nociceptin was not due to tachyphylaxis because themaximally effective concentration of nociceptin (0.31 interval between the microinjections of nociceptin was

96 N. Shah et al. / Brain Research 984 (2003) 93–103

Fig. 1. Effect of vagotomy on HR responses to nociceptin. In each panel, in this and other figures, the top trace represents pulsatile arterial pressure(PAP;mmHg), second trace represents mean arterial pressure (MAP, mmHg) and the bottom trace represents HR (bpm). (A) nCom was identified by amicroinjection ofL-Glu (5 mM); usual pressor and tachycardic responses were elicited; (B) 5 min later, nociceptin (0.31 mM) was microinjected at thesame site; depressor and bradycardic responses were elicited; (C) 20 min later, the right vagus nerve was sectioned. After allowing a 2-min period forstabilization, nociceptin (0.31 mM) was again injected at the same site; unilateral vagotomy did not alter nociceptin-induced responses; (D) 20 minlaterthe second vagus nerve was sectioned; 2 min later, nociceptin (0.31 mM) was again injected at the same site; bilateral vagotomy abolished bradycardiaandattenuated depressor responses elicited by nociceptin.

more than 20 min. The ORL1 receptor antagonist did not 3 .3. Effect of ORL1 receptor antagonist on L-Glualter the pressor and tachycardic responses elicited byresponsesL-Glu (5 mM) microinjections at the same site (Fig. 2E;see also Section 3.3). The blocking effect of the ORL1 These experiments were done in the same group of ratsreceptor antagonist lasted for 40–60 min.Fig. 2F shows that was used in Section 3.2 (n59). The possibility that

1the recovery of nociceptin responses. The blocking effect microinjections of [N-Phe ]–nociceptin-(1–13)–NH (4.52

of ORL1 receptor antagonist on nociceptin-induced re- mM) into the nCom may exert deleterious neuronal effectssponses is shown graphically inFig. 3. In these experi- at the site of injection, which may explain the lack ofments (n59), the decreases in MAP in response to responses to nociceptin, was explored as follows.L-Glu (5microinjections of nociceptin (0.31 mM) into the nCom mM) was microinjected into the nCom before and after thebefore and after the microinjections of the ORL1 receptor microinjection of the ORL1 receptor antagonist (4.5 mM).antagonist (4.5 mM) were 32.262.1 and 0.161.5 mmHg, Microinjections of the ORL1 receptor antagonist did notrespectively (P,0.01) (Fig. 3A). Likewise, the decreases attenuateL-Glu-induced responses. For example, theL-in HR in response to microinjections of nociceptin (0.31 Glu-induced increases in MAP before and after the mi-mM) into the nCom before and after microinjections of the croinjection of the ORL1 receptor antagonist (4.5 mM)ORL1 receptor antagonist (4.5 mM) were 42.266.8 and were 24.463.8 and 23.964 mmHg, respectively (P.0.05)1.161.4 bpm, respectively (P,0.01) (Fig. 3B). Concen- (Fig. 3C). The HR increases elicited by microinjections oftrations of the ORL1 receptor antagonist less than 4.5 mM L-Glu before and after the microinjections of the ORL1did not block the effects of 0.31 mM nociceptin. For receptor antagonist (4.5 mM) were 29.464.9 andexample, the decreases in MAP induced by nociceptin 25.665.5 bpm, respectively (P.0.05) (Fig. 3D).(0.31 mM) before and after the microinjection of a smallerconcentration of the ORL1 receptor antagonist (2.5 mM)were 3865 and 35610 mmHg, respectively (P.0.05) 3 .4. Effect of ORL1 receptor antagonist on(n54). In the same group of rats, the decreases in HR endomorphin-2 responsesinduced by nociceptin (0.31 mM) before and after the

1microinjection of ORL1 receptor antagonist (2.5 mM) The selectivity of [N-Phe ]–nociceptin-(1–13)–NH as2

were 4068 and 4565 bpm, respectively (P.0.05). an antagonist for ORL1 opioid receptors was determined

N. Shah et al. / Brain Research 984 (2003) 93–103 97

Fig. 2. Blockade of nociceptin responses in the nCom. (A) Pressor and tachycardic responses to microinjection ofL-Glu (5 mM); (B) after allowing aninterval of 5 min between injections, nociceptin (0.31 mM) was microinjected at the same site; depressor and bradycardic responses were elicited; (C) a

120-min interval was allowed to avoid tachyphylaxis to nociceptin responses and [N-Phe ]–nociceptin-(1–13)–NH (ORL1 receptor antagonist; 4.5 mM)2

was microinjected at the same site. The antagonist did not elicit any response by itself; (D) a 2-min interval allowed the diffusion of the antagonist into thenCom. Then, nociceptin (0.31 mM) was microinjected at the same site; the responses to nociceptin were blocked; (E) 5 min later,L-Glu (5 mM) wasmicroinjected at the same site; usual pressor and tachycardic responses were elicited; (F) the blocking effect of the ORL1 receptor antagonist lasted for40–60 min; recovery of nociceptin responses is shown in this panel.

as follows. The nCom was identified by microinjections of injection of the antagonist were similar (P.0.05) (Fig.L-Glu (5 mM); usual pressor and tachycardic responses 4E).were elicited (Fig. 4A). After an interval of 5 min,endomorphin-2 (0.6 mM) was microinjected at the same 3 .5. Effect of naloxone on endomorphin-2 responsessite, and decreases in MAP and HR were observed whichlasted for 5–6 min (Fig. 4B). The dose of endomorphin-2 Microinjections ofL-Glu (5 mM) were used to identify(0.6 mM) was selected on the basis of our preliminary the nCom from which the pressor and tachycardic re-studies[48]. At 20 min after the BP and HR returned to sponses were elicited (Fig. 5A). After an interval of 5 min,basal levels, the ORL1 receptor antagonist (4.5 mM) was endomorphin-2 (0.6 mM) was microinjected at the samemicroinjected at the same site. After allowing a short time site, and decreases in MAP and HR were observed which(2 min) for the antagonist to diffuse into the nCom, lasted for 5–6 min (Fig. 5B). At 20 min after the BP andendomorphin-2 (0.6 mM) was again injected at the same HR returned to basal levels, naloxone (0.125 mM) wassite. The ORL1 receptor antagonist did not alter the microinjected at the same site. In this and other series ofresponses to endomorphin-2 (Fig. 4C). These results are experiments, microinjections of naloxone alone did notshown graphically inFig. 4D and E.The decreases in elicit any cardiovascular response. After allowing a 2-minMAP induced by endomorphin-2 (0.6 mM) before and period for naloxone to diffuse into the nCom,after the injection of the ORL1 receptor antagonist (4.5 endomorphin-2 (0.6 mM) was again injected at the samemM) were not statistically different (21.161.8 mmHg) site; the cardiovascular responses to endomorphin-2 were(n59) (P.0.05) (Fig. 4D). In the same group of rats, the completely blocked (Fig. 5C). The lack of responses todecreases in HR induced by endomorphin-2 (0.6 mM) endomorphin-2 was not due to tachyphylaxis because abefore (23.363.3 bpm) and after (21.163.1 bpm) the period of more than 20 min was allowed between the two

98 N. Shah et al. / Brain Research 984 (2003) 93–103

were elicited (Fig. 6A). After an interval of 5 min,nociceptin (0.31 mM) was microinjected at the same site,and decreases in MAP and HR were observed which lastedfor 3.1–5.7 min (Fig. 6B). At 20 min after the BP and HRreturned to basal levels, naloxone (0.125 mM) was mi-croinjected at the same site. After allowing a period of 2min for naloxone to diffuse into the nCom, nociceptin(0.31 mM) was again injected at the same site; thecardiovascular responses to nociceptin were not alteredsignificantly (Fig. 6C). These results are shown graphicallyin Fig. 6D and E.In these experiments, the decreases inMAP in response to microinjections of nociceptin (0.31mM) into the nCom before (33.663.7 mmHg) and after(35.764.6 mmHg) the microinjections of naloxone (0.125mM) were not significantly different (P.0.05) (Fig. 6D).In the same group of rats, the decreases in HR in responseto microinjections of nociceptin (0.31 mM) into the nCombefore (37.966.3 bpm) and after (37.164.6 bpm) themicroinjections of naloxone (0.125 mM) were not sig-nificantly different (P.0.05) (Fig. 6E).

Fig. 3. Graphic representation of the effects of the ORL1 receptor3 .7. Effect of naloxone on L-Glu responsesantagonist. (A) The depressor responses to microinjections of nociceptin

(0.31 mM) into the nCom before (32.262.1 mmHg) and after (0.161.51mmHg) the microinjections of [N-Phe ]–nociceptin-(1–13)–NH (4.52 The possibility that microinjections of naloxone (0.125

mM) at the same site were significantly different (*,P,0.01). (B) The mM) into the nCom may exert deleterious neuronal effectsbradycardic responses to microinjections of nociceptin before (42.266.8

at the site of injection that may explain the lack of effect ofbpm) and after (1.161.4 bpm) the microinjections of the antagonist wereendomorphin-2 after the injection of naloxone was investi-significantly different (*, P,0.01). (C) The pressor responses to mi-

croinjections of L-Glu (5 mM) into the nCom before and after the gated in the same group of rats used in Section 3.5 (n57).1microinjections of [N-Phe ]–nociceptin-(1–13)–NH (4.5 mM) at the2 L-Glu (5 mM) was microinjected into the nCom before and

same site, 24.463.8 and 23.964 mmHg, respectively, were not sig- after the microinjection of naloxone. Microinjections of thenificantly different (P.0.05). (D) The tachycardic responses to mi-

naloxone did not attenuateL-Glu-induced responses. Forcroinjections ofL-Glu before (29.463.8 bpm) and after (25.665.5 bpm)example, theL-Glu-induced increases in MAP before andthe microinjections of the ORL1 receptor antagonist were also not

significantly different (P.0.05) (n59). after the microinjections of naloxone (0.125 mM) were23.864.3 and 26.363.8 mmHg, respectively (P.0.05). Inthe same rats, the increases in HR responses to microinjec-

injections of endomorphin-2. These results are showntions of L-Glu before and after the microinjections of

graphically inFig. 5D and E.In these experiments (n57),naloxone were 26.363.8 and 21.366.6 bpm, respectively

the decreases in MAP in response to microinjections of(P.0.05).

endomorphin-2 (0.6 mM) into the nCom before (25.762.5mmHg) and after (0.760.8 mmHg) the microinjections ofnaloxone (0.125 mM) were significantly different (P, 3 .8. Histology0.01) (Fig. 5D). In the same group of rats, the decreases inHR in response to microinjections of endomorphin-2 (0.6 A typical site of microinjection in the nCom is shown inmM) into the nCom before (31.463.4 bpm) and after Fig. 7A. The center of the site was located 0.3 mm caudal(4.364.2 bpm) the microinjections of naloxone (0.125 to the calamus scriptorius, 0 mm lateral to the midline andmM) were also significantly different (P,0.01) (Fig. 5E). 0.3 mm deep from the dorsal surface of the brain. InFig.

7B, the sites of microinjections into the nCom in different3 .6. Effect of naloxone on nociceptin responses rats (n55) were marked on the same section. The selection

of rats for the histological studies included three rats inAlthough naloxone has been reported to blockm, d and which a maximally effective concentration of nociceptin

k opioid receptors[45,49], it is ineffective at ORL1 opioid was microinjected (Section 3.1) and two rats in which thereceptor [7,8]. Therefore, the effect of naloxone on blockade of nociceptin responses by prior microinjectionsnociceptin-induced responses in the nCom was tested in of the ORL1 receptor antagonist was tested (Section 3.2).the same group of rats used in Section 3.5 (n57). The sites of microinjection were located 0.3–0.4 mmMicroinjections ofL-Glu (5 mM) were used to identify the caudal to the CS, 0–0.2 mm lateral to the midline andnCom from which the pressor and tachycardic responses 0.3–0.4 mm deep from the dorsal medullary surface.

N. Shah et al. / Brain Research 984 (2003) 93–103 99

Fig. 4. Selectivity of ORL1 receptor antagonist. (A) Microinjection ofL-Glu (5 mM) into the nCom elicited usual pressor and tachycardic responses; (B) 51min later, a microinjection of endomorphin-2 (0.6 mM) at the same site elicited depressor and bradycardic responses; 20 min later [N-Phe ]-endomorphin-

2-(1–13)–NH (4.5 mM) was microinjected at the same site (large arrow between B and C); no cardiovascular responses were elicited (not shown); (C) 22

min later, endomorphin-2 (0.6 mM) was again microinjected at the same site; the ORL1 opioid receptor antagonist did not alter the responses toendomorphin-2; (D) bar graph showing that endomorphin-2-induced depressor responses were not altered by the ORL1 receptor antagonist; the depressor

1responses before and after the microinjections of [N-Phe ]–nociceptin-(1–13)–NH (4.5 mM) (2161.8 mmHg) were identical; (E) endomorphin-2-2

induced bradycardic responses were not altered by the ORL1 receptor antagonist; the bradycardic responses before and after the microinjections of theORL1 receptor antagonist (4.5 mM), 23.363.3 and 21.163.1 bpm, respectively, were not significantly different (P.0.05) (n59).

Fig. 5. Blockade of endomorphin-2 responses. (A) Identification of the nCom by a microinjection ofL-Glu (5 mM); (B) 5 min later, endomorphin-2 (0.6mM) was microinjected at the same site; depressor and bradycardic responses were elicited. At 20 min later, naloxone (0.125 mM) was microinjected atthe same site (large arrow between B and C); no cardiovascular responses were elicited by naloxone alone (not shown); (C) depressor and bradycardicresponses to microinjection of endomorphin-2 (0.6 mM) were blocked by naloxone (0.125 mM) microinjected into the nCom two min prior to the injectionof endomorphin-2; (D) depressor responses to microinjections of endomorphin-2 (0.6 mM) before (25.762.5 mmHg) and after (0.760.8 mmHg) themicroinjections of naloxone (0.125 mM) were significantly different (*,P,0.01) (n57). (E) In the same group of rats, the bradycardic responses tomicroinjections of endomorphin-2 before (31.463.4 bpm) and after (4.364.2 bpm) the microinjections of naloxone were significantly different (*,P,0.01).

100 N. Shah et al. / Brain Research 984 (2003) 93–103

Fig. 6. Naloxone does not block the effects of nociceptin. (A) Identification of nCom site with a microinjection ofL-Glu (5 mM); (B) 5 min later, amicroinjection of nociceptin (0.31 mM) elicited usual depressor and bradycardic responses; 20 min later, naloxone (0.125 mM) was microinjected at thesame site (large arrow between B and C); (C) 2 min later, nociceptin (0.31 mM) was microinjected at the same site; the depressor and bradycardicresponses to nociceptin remained unaltered; (D) depressor responses before (33.663.7 mmHg) and after (35.764.6 mmHg) the microinjections ofnaloxone (0.125 mM) were not significantly different (P.0.05) (n57); (E) in the same group of rats, the bradycardic responses before (37.966.3 bpm)and after (37.164.6 bpm) the microinjections of the naloxone (0.125 mM) were also not significantly different (P.0.05).

4 . Discussion with the effects of nociceptin in the nCom for comparisonof our results. As stated earlier, the presence of ORL1

In this study microinjections ofL-Glu were used to receptors in the commissural subnucleus of the nTS hasfunctionally identify the nCom. As stated earlier,L-Glu is been demonstrated in several reports[1,20,31,37,41].known to excite neuronal cell bodies but not fibers of Microinjections of nociceptin may act on ORL1 receptorspassage[21]. Consistent with our earlier reports, excitation in the nCom and exert an inhibitory effect on theof nCom neurons byL-Glu resulted in an increase in BP chemosensitive nCom neurons. In this context, it may beand HR [56]. Based on the current knowledge regarding noted that at the cellular level, ORL1 receptors have beenthe organization of medullo-spinal cardiovascular areas, demonstrated to act through the intracellular mechanismsthe mechanism by whichL-Glu exerts these effects can be similar to those utilized bym, d and k opioid receptors.postulated as follows[47]. We have previously reported These mechanisms include: (1) inhibition of adenylatethat chemosensitive neurons are present in the general area cyclase[36,46], (2) activation of potassium channels[14],encompassing the commissural subnucleus of nTS[11]. and/or (3) inhibition of calcium channels[13,15]. Thus,These neurons have been reported to project to the RVLM direct postsynaptic effect of nociceptin on neurons isand glutamate has been implicated as a neurotransmitter in usually inhibitory[4,30,57].Nociceptin-induced inhibitionthis projection [29,52]. Thus, activation of glutamate of nCom neurons may decrease their excitatory input to thereceptors in the nCom may result in the activation of RVLM-neurons. Consequently, a decrease in the activity ofRVLM-neurons via excitatory amino acid receptors. RVLM-neurons may decrease the excitatory input toStimulation of the RVLM-neurons increases the excitatory sympathetic preganglionic neurons located in the IMLinput to sympathetic preganglionic neurons located in the causing depressor responses. Since the heart receives bothintermediolateral cell column of the spinal cord (IML) and sympathetic and parasympathetic innervation, a decrease inpressor and tachycardic responses are elicited[53]. the activity of sympathetic innervation to the heart would

Our results indicate that microinjections of nociceptin result in a predominance of parasympathetic vagal effectsinto the nCom elicit depressor and bradycardic responses. on the heart thus causing a bradycardia. This may explainThese effects lasted for 5–6 min; peptidases have been why bilateral vagotomy abolished the bradycardic re-implicated in the termination of nociceptin effects[54]. sponses to microinjections of nociceptin into the nCom. AAlthough, cardiovascular responses to microinjections of significant reduction in the depressor responses to nocicep-nociceptin into the nTS[33] and RVLM have been studied tin was also noted after bilateral vagotomy. This observa-[27], there is no detailed report in the literature dealing tion suggests that the bradycardia induced by microinjec-

N. Shah et al. / Brain Research 984 (2003) 93–103 101

microinjections of nociceptin into the nCom appear to besite specific because similar microinjections into an adja-cent nTS region (approximately 0.5 mm rostral to thecalamus scriptorius, 0.5 mm lateral to the midline and 0.5mm deep from the medullary dorsal surface) elicit pressorand tachycardic responses[33].

In the present investigation, no tachyphylaxis wasobserved when the interval between microinjections ofnociceptin was at least 20 min. This observation is inagreement with reports in which no desensitization ofORL1/OP receptors was observed[51]. It should be4

noted, however, that desensitization of ORL1/OP re-4

ceptors has been reported in some in vitro studies[25].1As mentioned in Section 3.2, [N-Phe ]–nociceptin-(1–

13)–NH has been reported to be a specific blocker of2

nociceptin effects[3,6,22].Our observation that the effects1of nociceptin in the nCom were blocked by [N-Phe ]–

nociceptin-(1–13)–NH are consistent with these reports.2

The specificity of this ORL1 receptor antagonist wasdemonstrated in our investigation by its lack of antagonismagainst endomorphin-2 (am-receptor agonist)[60]. Fur-thermore, naloxone, which blocked the effects ofendomorphin-2 in the nCom, was ineffective against theeffects of nociceptin in this region. This observation isconsistent with earlier reports in which naloxone wasfound to be ineffective against nociceptin[7,8]. Microin-

1jections of [N-Phe ]–nociceptin-(1–13)–NH or naloxone2

alone into the nCom did not elicit any response indicatingFig. 7. (A) Photograph of a coronal medullary section (0.3 mm caudal to

that neither ORL1 receptors nor other classical opioidthe calamus scriptorius) showing a typical microinjection-site in thereceptors (m, d and k opioid receptors) in this region arenCom marked by India ink (30 nl). The center of the site of microinjec-involved in tonic control of cardiovascular function. Thetion in this section was located in the midline and 0.3 mm deep from the

1dorsal medullary surface; (B) the same photograph was used to show theconcentrations of [N-Phe ]–nociceptin-(1–13)–NH and2center of the microinjection sites (marked by a dark spot) observed in naloxone that blocked the effects of nociceptin andother animals (n55). Each spot represents one animal.

endomorphin-2, respectively, did not attenuate the re-sponses toL-Glu when microinjected into the nCom

tions of nociceptin into the nCom was partially responsible indicating that no deleterious effects were exerted by eitherfor the magnitude of the decrease in BP induced by these of these antagonists on the nCom neurons. Therefore, the

1microinjections. blockade of the responses to nociceptin by [N-Phe ]–There may be another explanation for the mechanism of nociceptin-(1–13)–NH , and endomorphin-2 by naloxone,2

depressor responses to microinjections of nociceptin into was in fact due to the blockade of ORL1 and other opioidthe nCom. The presence of presynaptic ORL /OP re- receptors, respectively.1 4

ceptors has been reported[32,58]. Activation of the The physiological implications of the present work canpresynaptic ORL1 receptors by nociceptin has been re- be only speculated at this time. It is hypothesized thatported to inhibit transmitter release (e.g. norepinephrine) nociceptin may serve as a neuromodulator in the neural[50]. Thus it is possible, that nociceptin acts on the circuits mediating cardiovascular reflexes. Since the mid-presynaptic ORL1 receptors causing an inhibition of line area in the nCom has been implicated in mediatingtransmitter release from afferent terminals. One source of carotid chemoreflex in the rat[10,11,56], this opioidthe afferent terminals in the nCom is the carotid body peptide may be released in this region in situations in[10,19] and these terminals are believed to be primarily which attenuation of cardiovascular reflex responses wouldglutamatergic[56]. Nociceptin has been reported to inhibit be desirable. For example, bradycardia is one of theglutamate release and glutamatergic neurotransmission in prominent responses to the activation of chemoreceptors.certain areas of the brain[18,42]. Reduction of glutamate In stressful situations profound bradycardia may be detri-release in the nCom and depression of glutamatergic mental to the individual because adequate cardiac functionneurotransmission in this region may, in turn, result in is required for an efficient perfusion of vital organs such asdepressor and bradycardic responses. the brain. Suppression of normal cardiovascular reflexes in

The depressor and bradycardic responses elicited by general and chemoreflex in particular under stressful

102 N. Shah et al. / Brain Research 984 (2003) 93–103

commissural subnucleus of nucleus tractus solitarius of the rat, Am.conditions may be beneficial to the individual under theseJ. Physiol. 268 (1995) R851–R858.circumstances[28,43,44].

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