Sympathoexcitatory neurotransmission of the chemoreflex ...€¦ · Sympathoexcitatory neurotransmission of the chemoreflex in the NTS of awake rats ANDREÁ S. HAIBARA,1 LENI G

Sympathoexcitatory neurotransmission of the chemoreflexin the NTS of awake rats

ANDREA S. HAIBARA,1 LENI G. H. BONAGAMBA,2 AND BENEDITO H. MACHADO2

1Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University ofMinas Gerais, 31270-901, Belo Horizonte, Minas Gerais; and 2Department of Physiology, School ofMedicine of Ribeirao Preto, University of Sao Paulo, 14049-900, Ribeirao Preto, Sao Paulo, Brazil

Haibara, Andrea S., Leni G. H. Bonagamba, andBenedito H. Machado. Sympathoexcitatory neurotransmis-sion of the chemoreflex in the NTS of awake rats. Am. J.Physiol. 276 (Regulatory Integrative Comp. Physiol. 45):R69–R80, 1999.—Cardiovascular responses to chemoreflexactivation by potassium cyanide (KCN, 20 µg/rat iv) wereanalyzed before and after the blockade of ionotropic ormetabotropic receptors into the nucleus of the solitary tract(NTS) of awake rats. Microinjection of ionotropic antagonists[6,7-dinitroquinoxaline-2,3-dione or kynurenic acid (Kyn)]into the lateral commissural NTS (NTSlat), the midlinecommissural NTS (NTSmid), or into both (NTSlat1mid), pro-duced a significant increase in basal mean arterial pressure,and the pressor response to chemoreflex activation was onlypartially reduced, whereas microinjection of Kyn into theNTSmid produced no changes in the pressor response to thechemoreflex. The bradycardic response to chemoreflex activa-tion was abolished by microinjection of Kyn into the NTSlat orinto NTSlat1mid but not by Kyn microinjection into the NTSmid.Microinjection of a-methyl-4-carboxyphenylglycine, a metabo-tropic receptor antagonist, into the NTSlat or NTSmid producedno changes in baseline mean arterial pressure or heart rate orin the chemoreflex responses. These results indicate that 1)the processing of the parasympathetic component (bradycar-dia) of the chemoreflex seems to be restricted to the NTSlatand was blocked by ionotropic antagonists and 2) the pressorresponse of the chemoreflex was only partially reduced bymicroinjection of ionotropic antagonists and not affected byinjection of metabotropic antagonists into the NTSlat orNTSmid or into NTSlat1mid in awake rats.

nucleus of the solitary tract; arterial chemoreceptors; cardio-vascular regulation; N-methyl-D-aspartate receptors;non-N-methyl-D-aspartate receptors; metabotropic receptors;kynurenic acid; 6,7-dinitroquinoxaline-2,3-dione; a-methyl-4-carboxyphenylglycine

THE NUCLEUS OF THE SOLITARY TRACT (NTS) is the mostimportant synaptic station in the brain stem thatintegrates afferent information from the cardiovascu-lar system producing the appropriate autonomic andventilatory adjustments required in different physiologi-cal situations. The medial and lateral commissuralsubnucleus of the NTS has been shown to be theprimary site of termination of cardiovascular afferentfibers, receiving inputs from carotid chemoreceptors,arterial baroreceptors, and cardiopulmonary receptors(4, 8, 17, 24, 27, 34).

The peripheral chemoreceptors are important in theregulation of respiratory and cardiovascular functions(8, 25), and the neurotransmission of the chemorecep-tors afferent in the NTS as well as in other areas of theventral medulla has been extensively studied (1, 20, 21,27, 28, 39). Pharmacological studies have indicatedthat excitatory amino acid (EAA) receptors in thecommissural NTS play an important role in the neuro-transmission of the peripheral chemoreflex (39–41).However, most of these experiments were performedwhile subjects were under anesthesia, which may havea distorting effect on the cardiovascular and ventilatoryresponses to chemoreflex activation (10). In addition,microinjection of L-glutamate into the NTS of consciousrats elicited opposite cardiovascular responses whencompared with the responses obtained in the sameanimals under anesthesia (23), further confirming thatneurotransmission in the NTS is deeply affected by theanesthesia.

In a previous study, we showed that bradycardicresponse induced by chemoreflex activation was medi-ated by N-methyl-D-aspartate (NMDA) receptors in thecommissural NTS because bilateral microinjection ofDL-2-amino-5-phosphonovaleric acid (AP-5), a selectiveNMDA receptor antagonist, into the lateral portion ofthe commissural NTS (NTSlat) blocked the bradycardicresponse in a dose-dependent manner. However, AP-5produced no effect on the pressor response to thechemoreflex, suggesting that the sympathoexcitatorycomponent of the chemoreflex may be mediated bynon-NMDA receptors (16). On the basis of such find-ings, the present study aimed to evaluate the role ofnon-NMDA receptors in the neurotransmission of thepressor response to chemoreflex activation (sympatho-excitatory component) in the lateral and medial aspectsof the commissural NTS.

Anatomic studies by Finley and Katz (9) have shownthat the projections of the carotid body afferents occurmainly in the NTSlat. Electrophysiological studies byMifflin (27) further confirmed that neurons in this areareceived input from carotid chemoreceptors. In con-trast, recent studies by Chitravanshi and colleagues(2, 3) reported that the carotid chemoreceptor afferentsseem to project mainly to the midline portion of thecommissural subnucleus of the NTS (NTSmid), at thecalamus scriptorium level. In addition, the blockade ofEAA receptors in the NTSmid of anesthetized ratsabolished the pressor and ventilatory responses tochemoreflex activation (39). Therefore, we also evalu-ated the role of EAA receptors (ionotropic and metabo-tropic) in the different subregions of the commissuralNTS (lateral and midline portions) in the neurotrans-

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mission of the sympathoexcitatory component of thechemoreflex (pressor response) in conscious rats. Apreliminary report of our findings has been publishedas an abstract (15).

METHODS

Guide cannula implantation in direction of NTS. MaleWistar rats weighing 230–270 g were used in the presentstudy. Four days before the experiments, the rats were deeplyanesthetized with tribromoethanol (250 mg/kg ip; Aldrich,Milwaukee, WI) and placed in a stereotaxic frame (Kopf,Tujunga, CA) for guide cannula implantation. When the ratreacted to frequent toe pinching during stereotaxic surgery,additional tribromoethanol was injected. The technique de-scribed by Michelini and Bonagamba (26) was adapted toimplant guide cannulas in the following three experimentalprotocols: 1) bilateral guide cannulas in the direction of theNTSlat (0.5 mm lateral to midline and ,0.5 mm rostral tocalamus scriptorium), 2) one guide cannula in direction of theNTSmid (at midline at level of calamus scriptorium), and 3)three guide cannulas, with two implanted in the direction ofthe NTSlat and one implanted in the direction of the NTSmid.The implants of all guide cannulas were performed in accor-dance with the coordinates of Paxinos and Watson (29). Toimplant each guide cannula, we made a small window in theskull caudal to the lambda and a 15-mm-long stainless steelguide cannula (22 gauge; Small Parts) was introduced perpen-dicularly through the window at the following coordinates:0.5 (NTSlat) or 0.0 mm (NTSmid) lateral to the bregma, 14.00(NTSlat) or 14.5 mm (NTSmid) caudal to the bregma, and 7.9mm below the skull surface at the bregma (NTSlat andNTSmid). The tip of the guide cannula was positioned ,1.0mm above the dorsal surface of the brain stem. The guidecannula was fixed to the skull with methacrylate and watchscrews and then closed with an occluder until time of experi-mentation.

Arterial and venous cannulation. One day before theexperiments, while rats were under tribromoethanol anesthe-sia, a catheter (PE-10 connected to PE-50; Clay Adams,Parsippany, NJ) was inserted into the abdominal aortathrough the femoral artery for measurement of pulsatilearterial pressure (PAP), mean arterial pressure (MAP), andheart rate (HR). A second catheter was inserted into thefemoral vein for systemic administration of potassium cya-nide (KCN). Both catheters were tunneled subcutaneouslyand exteriorized through the back of the neck to be connectedto the pressure transducer on the subsequent day. PAP andMAP were measured in conscious, freely moving rats with apressure transducer (model CDX III; Cobe, Lakewood, CO)connected to a Narcotrace 80 physiological recorder (NarcoBio-Systems, Austin, TX). HR was measured with a NarcoBiotachometer Coupler (model 7302).

Microinjections into NTS. For microinjections into theNTS, a 33-gauge needle (Small Parts) 1.5 mm longer than theguide cannula was connected by PE-10 tubing to a 1-µlsyringe (Hamilton, Reno, NV). After removal of the occluder,the needle for microinjection was carefully inserted into theguide cannula and manual injection was started 30 s later.For bilateral microinjection, the microinjection was initiallyperformed on one side, the needle was withdrawn andrepositioned in the contralateral side, and the second injec-tion was performed. The same procedure was used in theprotocols with three guide cannulas. Therefore, the timeinterval of microinjections into the NTS in the differentprotocols was ,1 min. The volume for each microinjectionwas 100 nl in all experimental protocols.

Activation of chemoreflex. The chemoreflex was activatedby intravenous injection of KCN (20 µg/rat; Merck, Darm-stadt, Germany) in accordance with the procedures describedby Franchini and Krieger (10) and was previously validatedfor our experimental conditions (16). These previous studiesdemonstrated that the cardiovascular responses to KCNinjection were reproducible, and no habituation was observedwhen the same dose of KCN was systematically injected atintervals of 10 min (16).

Drugs. The drugs microinjected into the NTS were dilutedin artificial cerebrospinal fluid (aCSF) containing (in mM) 3KCl, 0.6 MgCl2, 2 CaCl2, 132 NaCl, 24 NaHCO3, and 4dextrose or 0.9% saline. The solutions were freshly dissolvedin aCSF, except 6,7-dinitroquinoxaline-2,3-dione (DNQX),which was dissolved in 2.5% DMSO (Sigma, St. Louis, MO).Sodium bicarbonate was added to the solutions to adjust thepH to a range of 7.0–7.4.

Experimental protocols. All studies were performed inconscious, freely moving animals. The experimental protocolfor the study of neurotransmission into the NTS consisted ofthe activation of the carotid chemoreflex before and aftermicroinjection of EAA antagonists into the NTSlat or NTSmidor into both sites (NTSlat1mid) by using three guide cannulas.The NTS was functionally identified by previous microinjec-tion of L-glutamate (1 nmol/100 nl), which, in accordance withour previous studies (5, 6, 23), produces pressor and brady-cardic responses of short duration. The peak changes in MAPand HR in response to chemoreflex activation were evaluatedbefore and 10 min after the microinjections of EAA receptorantagonists into the NTS. A third injection of KCN wasperformed 40 min after antagonist microinjection to evaluatethe reversibility of the blockade. The EAA receptor antago-nists microinjected into the NTS were DNQX (ResearchBiochemicals International, Natick, MA), a selective non-NMDA receptor antagonist, in three different doses (0.1, 0.5,and 2.0 nmol/100 nl), kynurenic acid (Kyn; Sigma), a nonselec-tive ionotropic antagonist, in one dose (10 nmol/100 nl), anda-methyl-4-carboxyphenylglycine (MCPG; Research Biochemi-cals International), a selective metabotropic antagonist, intwo different doses (2.5 and 5.0 nmol/100 nl). Each rat used inthe three different experimental protocols (DNQX, Kyn, orMCPG) received only one dose of the antagonist. The groupsof rats that received microinjection of EAA receptor antago-nists into the NTS also received a control microinjection ofsaline with DMSO (DNQX group) or aCSF (Kyn and MCPGgroups) in each individual experiment at least 30 min previ-ous to the microinjection of the respective antagonist.

The selectivity of DNQX for non-NMDA receptors wasevaluated in a specific protocol in which NMDA, a selectiveNMDA receptor agonist (10 pmol/100 nl), was microinjectedinto the NTS before and after different doses of DNQX, andthe effectiveness of MCPG in blocking metabotropic receptorswas tested in a specific protocol in which trans-(6)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD; Research Bio-chemicals International), a selective metabotropic receptoragonist (250 pmol/100 nl), was microinjected into the NTSbefore and after microinjection of MCPG (2.5 and 5.0 nmol/100 nl, respectively).

Histological examination. At the end of the experiments,100 nl of Evans blue dye (2%) were microinjected for histologi-cal identification of the sites of microinjection, and later theanimals were submitted to intracardiac perfusion with salinefollowed by 10% buffered Formalin while they were underether anesthesia. The brains were removed and stored inbuffered Formalin for 2 days. Serial coronal sections (10–15 µm thick) were obtained and stained by the Nissl method.Only the rats in which the microinjection sites were located in

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the lateral or midline or in both portions of the commissuralNTS, in accordance with the protocol, were considered fordata analysis.

Data analysis. All data are expressed as means 6 SE. Theresults were analyzed by one-way ANOVA, and the differ-ences between individual means were determined by Stu-dent’s t-test, with the level of significance set at 0.05 in allanalyses.

RESULTS

Effect of bilateral microinjection of DNQX into NTSlaton cardiovascular responses to chemoreflex activation.Figure 1 illustrates the effects of bilateral microinjec-tion of increasing doses of DNQX (0.1, 0.5, and 2.0nmol/100 nl) into the NTSlat on the cardiovascularresponses to chemoreflex activation with KCN (20 µgiv) of three different rats, representative of their respec-tive groups. Figure 1A shows that DNQX (0.1 nmol/100nl) produced no changes in the pressor or bradycardicresponses to chemoreflex activation, whereas the doseof 0.5 nmol/100 nl (Fig. 1B) attenuated the pressorresponse but produced no change in the bradycardicresponse induced by chemoreflex activation. Figure 1Cshows that the dose of 2.0 nmol/100 nl also attenuatedthe pressor response to the chemoreflex in the sameway as observed with the dose of 0.5 nmol/100 nl andblocked the bradycardic response. Bilateral microinjec-

tion of DNQX into the NTS produced a significantincrease in basal MAP. Table 1 shows the absolutevalues of MAP and HR before and 10 min after bilateralmicroinjection of DNQX into the NTSlat and indicatesthat DNQX produced a significant increase in basalMAP at all doses, without any significant changes inbasal HR.

The data related to the effects of DNQX on thechemoreflex responses are summarized in Fig. 2 andshow that increasing doses of DNQX reduced but didnot abolish the pressor response to the chemoreflexactivation when compared with the control response

Fig. 1. Changes in heart rate (HR), pulsatile arterial pressure (PAP), and mean arterial pressure (MAP) in responseto KCN (20 µg·0.1 ml21 ·rat21 iv) before and 10 min after bilateral microinjection of increasing doses of6,7-dinitroquinoxaline-2,3-dione (DNQX) [0.1 (A), 0.5 (B), and 2.0 nmol/100 nl (C)] into lateral portion of thecommissural nucleus of the solitary tract (NTSlat) of 3 different rats representative of their respective groups. bpm,Beats/min.

Table 1. Baseline MAP and HR before and 10 minafter bilateral microinjection of DNQX into NTSlat

DNQX,nmol/100 nl n

MAP, mmHg HR, beats/min

Before After Before After

0.1 7 9964 11162* 33967 3316100.5 5 11466 13967* 373615 390662.0 6 10065 12665* 338612 342612

Values are means 6 SE. MAP, mean arterial pressure; HR, heartrate; NTSlat, lateral portion of the commissural nucleus of the solitarytract. *Statistically different in relation to values before microinjec-tion of 6,7-dinitroquinoxaline-2,3-dione (DNQX) (P,0.05, pairedt-test).

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(155 6 2 vs. 154 6 4, 164 6 4 vs. 134 6 7, and 159 64 vs. 133 6 10 mmHg for doses of 0.1, 0.5, and 2.0nmol/100 nl, respectively) in the three groups of ratsstudied. In relation to the parasympathetic componentof the chemoreflex, the microinjection of DNQX at thedoses of 0.1 and 0.5 nmol/100 nl induced no significantchange in the bradycardic response (2173 6 16 vs.2153 6 28 and 2216 6 27 vs. 216 6 27 beats/min).However, at the dose of 2.0 nmol/100 nl, DNQX eliciteda significant reduction in this response (2160 6 25 vs.250 6 31 beats/min). The effects of DNQX on thechemoreflex responses were reversible, considering that40 min after bilateral microinjection of DNQX into theNTSlat, the chemoreflex responses were back to controlvalues. Bilateral microinjection of vehicle containing2.5% DMSO into the NTSlat was associated with negli-gible effects on the pressor and bradycardic responsesto chemoreflex activation (Table 2).

The effects of DNQX (0.5 nmol/100 nl) on the cardio-vascular responses to chemoreflex activation were re-stricted to the NTS, considering that misplaced micro-injections of DNQX into areas adjacent to the NTS didnot modify the pressor or bradycardic responses to thechemoreflex activation (Table 3).

The selectivity of the antagonist DNQX on non-NMDA receptors was tested by a specific protocol inwhich the cardiovascular responses induced by microin-

jection of the agonist NMDA (10 pmol/100 nl) into theNTS were evaluated before and after microinjection ofDNQX (0.5 or 2.0 nmol/100 nl) into the same site. Ta-ble 4 shows that DNQX at the dose of 0.5 nmol/100 nlproduced no significant changes in the depressor orbradycardic responses induced by the NMDA microin-jection, indicating that NMDA receptors are not af-fected by this dose of DNQX. However, the dose of 2.0nmol/100 nl of DNQX blocked the depressor and brady-cardic responses induced by the NMDA agonist, show-ing that this dose of DNQX also affects NMDA recep-tors.

Effect of microinjection of Kyn into NTSlat or NTSmidor into NTSlat1mid on cardiovascular responses to chemo-reflex activation. The effect of microinjection of Kyn intothe NTSlat or NTSmid on the cardiovascular responses tochemoreflex activation is illustrated in Fig. 3. Fig-ure 3A shows a tracing of one rat in which bilateralmicroinjection of Kyn (10 nmol/100 nl) into the NTSlatattenuated the pressor response to the chemoreflex,whereas the bradycardic response was abolished. Fig-ure 3B shows the tracings of one rat in which microin-jection of Kyn into the NTSmid induced small changes inthe pressor or bradycardic responses elicited by KCNinjection despite a significant increase in basal MAP.Figure 3C shows the tracings of one rat in whichsimultaneous microinjection of Kyn into NTSlat1midattenuated the pressor response and abolished thebradycardic response to chemoreflex similar to themicroinjection of Kyn into the NTSlat (Fig. 3A). Thesedata are summarized in Fig. 4 and show that Kynmicroinjected into the NTSlat or into the NTSlat1mid

Fig. 2. Changes (D) in MAP (top) and HR (bottom) in response toinjection of KCN (20 µg·0.1 ml21 ·rat21 iv), before (control) and 10min after bilateral microinjection of increasing doses of DNQX [0.1(s, n 5 7), 0.5 (n, n 5 5), and 2.0 nmol/100 nl (r, n 5 6)] into NTSlat in3 groups of rats. Top: significant differences in pressor response wereobserved after doses of 0.5 and 2.0 nmol/100 nl in comparison withcontrol. Bottom: changes in HR were significantly reduced only afterDNQX at 2.0 nmol/100 nl, a dose that is not selective for non-NMDAreceptors (Table 4). One-way ANOVA and differences between indi-vidual means were determined by Student’s modified t-test withBonferroni correction for multiple comparisons.

Table 2. Changes in MAP and HR in response tochemoreflex activation before and 10 min afterbilateral microinjection of 2.5% DMSO into NTSlat

DMSO (2.5%),nmol/100 nl n

D MAP, mmHg D HR, beats/min


0.1 7 15062 15261 2171621 21736210.5 5 16464 16363 2224628 22186332.0 6 15664 15664 2162627 2163629

Values are means 6 SE. No statistical difference in change in (D)MAP or D HR was observed when values before and after DMSOmicroinjection were compared (paired t-test). Injections were made insame group of rats that received microinjection of DNQX (0.1, 0.5,and 2.0 nmol/100 nl).

Table 3. Changes in MAP and HR in response tochemoreflex activation before and 10 min aftermisplaced microinjections of DNQX or Kyninto areas adjacent to NTS

n



DNQX,0.5 nmol/100 nl 3 15769 15367 2130635 2127630

Kyn, 10 nmol/100 nl 4 15562 14964 2175617 2160623

Values are means 6 SE. Kyn, kynurenic acid. No statisticaldifference in D MAP or D HR was observed when values before andafter misplaced microinjections were compared (paired t-test).



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significantly reduced the cardiovascular responses tochemoreflex activation. In these cases, the pressorresponses were significantly reduced (NTSlat, 150 6 2vs. 130 6 3 mmHg; NTSlat1mid, 148 6 3 vs. 122 6 8mmHg) and the bradycardic responses were almostabolished (NTSlat, 2225 6 28 vs. 218 6 7 beats/min;NTSlat1mid, 2213 6 28 vs. 211 6 5 beats/min). Incontrast, microinjection of Kyn into the NTSmid did notaffect the pressor (157 6 5 vs. 39 6 8 mmHg) orbradycardic response (2191 6 37 vs. 2163 6 64beats/min) to chemoreflex activation. Microinjection of

Kyn into the NTSlat, NTSmid, or NTSlat1mid induced asignificant increase in baseline MAP but not in baselineHR (Table 5).

The effects of Kyn microinjection into the NTSlat orNTSlat1mid on the chemoreflex were reversible, consider-ing that 60 min after microinjection of the antagonist,the cardiovascular responses to chemoreflex activationwere similar to those observed before microinjection. Inaddition, bilateral microinjection of the vehicle (aCSF)into the NTSlat (n 5 6) did not modify the pressor(148 6 3 vs. 151 6 1 mmHg) or bradycardic (2214 626 vs. 2216 6 27 beats/min) responses to chemoreflexactivation.

The effects of Kyn on the cardiovascular responses tochemoreflex activation were restricted to the NTS,because misplaced microinjections into areas adjacentto the NTS did not modify the pressor or bradycardicresponses to chemoreflex activation (Table 3).

Effect of microinjection of MCPG into NTSlat orNTSmid on cardiovascular responses to chemoreflex acti-vation. Neither dose of MCPG (2.5 and 5.0 nmol/100 nl)modified the cardiovascular responses to the chemore-flex activation. Thus only the data for the dose of 2.5

Fig. 3. Changes in PAP, MAP, and HR in re-sponse to KCN (20 µg·0.1 ml21 ·rat21 iv) beforeand 10 min after bilateral microinjection of kyn-urenic acid (Kyn, 10 nmol/100 nl) into NTSlat (A),midline portion of the commissural NTS (NTSmid,B), or NTSlat1mid (C) of 3 different rats represen-tative of their respective groups.

Table 4. Changes in MAP and HR produced by NMDAmicroinjection into NTS before and 10 minafter microinjection of DNQX into NTSlat

DNQX(nmol/100 nl) n



0.5 4 261619 248613 2145622 2856422.0 5 24769 2562* 2160625 2462*

Values are means 6 SE. NMDA (10 pmol/100 nl), N-methyl-D-aspartate. *Statistically different in relation to control values beforemicroinjection (P,0.05, paired t-test).



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nmol/100 nl are presented in Fig. 5, which illustratesthe effects of microinjections of MCPG into the NTSlator into the NTSmid on the cardiovascular responses tochemoreflex activation. Figure 5A shows the tracings ofone rat in which bilateral microinjection of MCPG intothe NTSlat induced no changes in the pressor or brady-cardic responses to chemoreflex activation, and Fig. 5Bshows the tracings of one rat in which microinjection ofMCPG into the NTSmid also did not affect the responsesto chemoreflex activation.

Figure 6 summarizes the data related to the blockadeof metabotropic receptors and shows that MCPG (2.5nmol/100 nl) microinjected into the NTSlat or NTSmiddid not modify the pressor response (NTSlat, 156 6 4 vs.153 6 6 mmHg; NTSmid, 145 6 8 vs. 145 6 9 mmHg)or in bradycardic response (NTSlat, 2241 6 18 vs.2226 6 16 beats/min; NTSmid, 2210 6 45 vs. 2212 6 45beats/min) to chemoreflex activation. Microinjection ofMCPG into these two subregions of the commissuralNTS did not elicit significant changes in baseline MAPor in baseline HR (Table 6).

The dose of MCPG microinjected into the NTSlat waseffective in blocking the metabotropic receptors, be-cause previous microinjection of MCPG (2.5 nmol/100nl) into the NTSlat of a specific group of rats (n 5 8)significantly reduced the depressor (266 6 8 vs. 224 69 mmHg) as well as the bradycardic (2267 6 26 vs.280 6 3 beats/min) responses induced by trans-ACPD(250 pmol/100 nl) microinjected into the same site.Similar blockade was achieved with the dose of 5.0nmol/100 nl of MCPG (data not shown).

Histological analyses of microinjection sites. Figure 7is a photomicrograph of a transverse section of thebrain stem of the rat, showing the site of microinjec-tions in the NTS. Figure 7A is a photomicrograph of acoronal section of the brain stem of one rat representa-tive of the group in which the microinjections wereperformed bilaterally into the NTSlat. Figure 7B is aphotomicrograph of a coronal section of the brain stemof one rat representative of the group in which themicroinjections were performed into the NTSmid.

Figure 8 is a schematic representation of the brainstem at the calamus scriptorium level and shows theoverlapping sites of Evans blue dye microinjections intothe NTSlat (Fig. 8A) or NTSmid (Fig. 8B) of rats used inthe protocols of Kyn microinjection into the NTS. Theaverage of the anterior-posterior extension of the areastained by Evans blue dye was 626 6 42 µm (NTSlat)and 648 6 89 µm (NTSmid).

DISCUSSION

Several lines of experimental evidence support theconcept that EAA receptors play a major role in theprocessing of cardiovascular afferents in the NTS (4,13, 14, 16, 38) and particularly in the neurotransmis-sion of the chemoreflex in the NTS (2, 3, 20, 27, 39, 40).In a recent study on unanesthetized rats, we showedthat the bradycardic response to chemoreflex activationwas blocked in a dose-dependent manner by AP-5, anNMDA receptor antagonist, whereas the pressor re-sponse was not affected (16). However, previous studiesperformed on anesthetized rats have reported thatadministration of EAAantagonists into the NTS blockedthe pressor response (sympathoexcitatory component)of the chemoreflex activation (39, 40); such blockadewas not observed in the present study in the absence ofanesthesia. Bilateral microinjection of DNQX (a selec-tive non-NMDA receptor antagonist) at the dose of 0.5nmol/100 nl, which does not affect NMDA receptors,significantly reduced but did not abolish the pressorresponse to chemoreflex activation (164 6 4 vs. 134 6

Fig. 4. Changes in MAP and HR in response to chemoreflex activa-tion with KCN (20 µg·0.1 ml21 ·rat21 iv), before (control) and 10 minafter microinjection of Kyn (10 nmol/100 nl) into NTSlat (n 5 6),NTSmid (n 5 6), or NTSlat1mid (n 5 6) of 3 different groups of rats.*Statistically different in relation to control response (P , 0.05,paired t-test).

Table 5. Baseline MAP and HR before and 10 minafter microinjection of Kyn (10 nmol/100 nl)into NTSlat, NTSmid, or NTSlat1mid

n



NTSlat 6 9963 12162* 380620 400620NTSmid 6 11164 12566* 377617 392618NTSlat1mid 6 10264 13664* 327612 32767

Values are means 6 SE. NTSmid, midline portion of the commis-sural NTS. *Statistically different in relation to values beforemicroinjection (P,0.05, paired t-test).



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17 mmHg), whereas the highest dose of DNQX used(2.0 nmol/100 nl), which is not selective for non-NMDAreceptors, did not elicit any additional reduction in thepressor response to chemoreflex activation (159 6 4 vs.133 6 10 mmHg).

The partial reduction of the pressor response tochemoreflex activation observed after DNQX may berelated, at least in part, to the involvement of non-

NMDA receptors in the neurotransmission of the sym-pathoexcitatory component of the chemoreflex in theNTSlat. However, these results require careful interpre-tation because another possibility to explain why thepressor response to chemoreflex activation was reducedby DNQX is the significant increase in the baselineMAP observed after the bilateral microinjection ofDNQX into the NTSlat. With respect to the DNQXprotocol, it is important to note that the additionalincrease in MAP in response to chemoreflex activationwas smaller than in the control, probably due to theincrease in the baseline sympathetic activity and conse-quently in MAP. It is also important to emphasize thatthe pressor response to chemoreflex activation is essen-tially due to sympathetic activation and not to vasopres-sin or any other circulating neurohormone, because inprevious studies we verified that the treatment withprazosin (intravenous), an a1-adrenoceptor antagonist,almost abolished the pressor response of the chemore-flex (16).

The effects observed after bilateral microinjection ofDNQX into the NTSlat on baseline MAP were probablydue to the blockade of the sympathoinhibitory pathwayof the baroreflex and suggest that non-NMDA receptorslocated on neurons of this subregion of the NTS are

Fig. 5. Changes in PAP, MAP, and HRin response to KCN (20 µg · 0.1ml21 ·rat21 iv) before and 5 min aftermicroinjection of a-methyl-4-carboxy-phenylglycine (MCPG; 2.5 nmol/100 nl)into NTSlat (A) or NTSmid (B) of 2 ratsrepresentative of their respectivegroups.

Fig. 6. Changes in MAP and HR in response to chemoreflex activa-tion with KCN (20 µg·0.1 ml21 ·rat21 iv) before (control) and 5 minafter microinjection of MCPG (2.5 nmol/100 nl) into NTSlat (n 5 8) orNTSmid (n 5 5) of 2 different groups of rats.

Table 6. Baseline MAP and HR before and 10 minafter microinjection of MCPG (2.5 nmol/100 nl)into NTSlat or NTSmid

n



NTSlat 8 10663 10664 381615 374617NTSmid 5 10262 10464 378627 382626

Values are means 6 SE. No statistical difference was observedwhen MAP and HR were compared before and after a-methyl-4-carboxyphenylglycine (MCPG) microinjection.



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involved in the neurotransmission of this component ofthe baroreflex. This possibility is supported by a previ-ous study by Gordon and Leone (12) showing thatnon-NMDA receptors in the NTS play a predominantrole in mediating the hypotensive response produced byaortic baroreceptor stimulation. In addition, we veri-fied in a previous study that the blockade of the NMDAreceptors in the NTSlat produced no changes in baselineMAP (16).

Another important aspect of the present study re-lates to the blockade of the bradycardic response tochemoreflex activation observed after the dose of 2.0nmol/100 nl of DNQX microinjected bilaterally into theNTSlat. In a previous study, we showed that the cardio-vagal component of this reflex is mediated by NMDAreceptors (16). Therefore, the bradycardic responseshould not be affected by the non-NMDA receptorantagonist unless the dose used is not selective for thisreceptor subtype. In this respect, a specific protocolused in the present study showed that DNQX at thedose of 2.0 nmol/100 nl is not selective for non-NMDAreceptors because it also blocked the cardiovascularresponses to NMDA microinjection into the NTS. Theprotocol using 0.5 nmol/100 nl of DNQX shows that thisdose is in the range of selectivity for non-NMDAreceptors, because the cardiovascular responses toNMDA receptors and the bradycardic response to che-moreflex activation were not affected, whereas baselineMAP was increased. Therefore, this evidence supportsthe concept that the cardiovagal component of thechemoreflex is effectively mediated by NMDA receptorsin the NTSlat.

The present data show that DNQX microinjected intothe NTSlat did not abolish the pressor response tochemoreflex activation. To explain these findings, atleast two possibilities should be considered. First, theEAA receptors in this subregion of the commissuralNTS, particularly the non-NMDA receptors, may not be

Fig. 7. Photomicrographs of transverse sections of brain stem show-ing bilateral microinjection sites in NTSlat at level of rostral edge ofarea postrema (A, NTSlat protocol) and site of microinjection intoNTSmid at level of calamus scriptorium (B, NTSmid protocol). Arrowsindicate sites of microinjections in NTS marked with Evans blue dye.

Fig. 8. Line drawing of transverse sec-tion of brain stem from 1 mm rostral to0.3 mm caudal to calamus scriptorium[adapted from Paxinos and Watson(29)]. Filled areas represent overlap-ping areas of Evans blue dye distribu-tion in NTSlat (A) or NTSmid (B) of allrats used in protocols of Kyn microinjec-tion into NTSlat or NTSmid. AP, areapostrema; Gr, gracile nucleus; Sol M,medial NTS; Sol C, commissural NTS;CC, central canal; X, dorsal motornucleus of vagus; XII, hypoglossalnucleus; NA, ambiguus nucleus; CVL,caudal ventrolateral medulla; Cu, cune-ate nucleus.



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involved in the sympathoexcitatory neurotransmissionof the chemoreflex. Second, the blockade of non-NMDAreceptors only in the NTSlat may not be sufficient toblock all the receptors located in different sites of theNTS probably involved in the processing of the afferentinformation for the chemoreflex. A study by Zhang andMifflin (40) showed that the pressor response of thechemoreflex was blocked when Kyn, a broad-spectrumEAA receptor antagonist, was microinjected into theNTSlat. In a study by Vardhan et al. (39), the pressorresponse to chemoreflex activation was also blockedonly after the blockade of NMDA and non-NMDAreceptors in the NTSmid. Therefore, in these studies(39, 40), which were performed in anesthetized ani-mals, the pressor response to chemoreflex activationwas abolished after a nonselective blockade of iono-tropic receptors in the NTSlat or NTSmid. Consideringthese previous studies and also that isolated blockadeof NMDA (16) or non-NMDA receptors (present study)in the NTSlat of unanesthetized rats was not able toblock the pressor response to chemoreflex activation,we also evaluated the effects of the Kyn microinjectioninto the NTSlat or NTSmid on the pressor response tochemoreflex activation in unanesthetized rats.

The reduction in magnitude of the pressor responseto chemoreflex activation induced by microinjection ofKyn into the NTSlat was similar to that induced bymicroinjection of DNQX into the same area. In anothergroup of rats, microinjection of Kyn into the NTSmid didnot affect the pressor response to chemoreflex activa-tion. In both cases (NTSlat and NTSmid), the microinjec-tion of Kyn produced a significant increase in basalMAP, similar to the effect of DNQX microinjection intothe NTSlat. One possibility that cannot be ruled out toexplain the reduction in the pressor response to chemo-reflex activation after microinjection of Kyn into NTSlatmay be related to the increase in baseline MAP, similarto that observed after microinjection of DNQX into theNTSlat. On the other hand, microinjection of Kyn intoNTSmid, despite increasing the baseline MAP, producedno attenuation in the pressor response to chemoreflexactivation. Therefore, this observation indicates thatthe reduction in the pressor response to chemoreflexactivation by Kyn microinjected into the NTS probablyis not associated with the increase in the baseline MAP.However, it is important to note that the baselineincreases in MAP produced by Kyn into the NTSlat orNTSmid were different (122 vs. 113%, respectively).This difference may explain why the increase in MAP inresponse to chemoreflex activation after Kyn into theNTSlat was smaller than the increase observed afterKyn into NTSmid. Similar findings were obtained withmicroinjections of DNQX into NTSlat. The dose of 0.1nmol/100 nl into the NTSlat increased baseline MAP by12% and produced no effect on the pressor response tochemoreflex activation, whereas the dose of 0.5 nmol/100 nl increased the baseline MAP by 22% and induceda significant reduction in the pressor response tochemoreflex activation. Alternatively, we might alsoconsider the possibility that the increase in baselineMAP, secondary to the basal increase in sympathetic

activity after Kyn or DNQX into the NTSlat, reduces themagnitude of the pressor response to chemoreflex acti-vation because the maximal increase of the sympa-thetic activity remained the same. The experimentalprotocol in which we performed blockade of EAA recep-tors in the NTSlat and NTSmid (NTSlat1mid) with Kyn (10nmol/100 nl) shows that the pressor response to chemo-reflex activation was also only partially reduced, sug-gesting that the processing of the excitatory componentof the chemoreflex in the NTS involves neurotransmit-ters and receptors other than EAAs and their receptors.

In relation to the parasympathetic component of thechemoreflex, our data show that Kyn microinjected intothe NTSlat abolished the bradycardic response, prob-ably due to the blockade of NMDA receptors, as wedemonstrated previously with microinjections of AP-5into the same subregion of the NTS (16). However, Kynmicroinjected into the NTSmid produced no change inthe bradycardic response to chemoreflex activation,suggesting that this subregion of the commissural NTSis not directly involved in the processing of the parasym-pathetic component of this reflex. A study by Colombariet al. (7) shows that the integrity of NTSmid is essentialfor the pressor and bradycardic responses to chemore-flex activation, but their findings do not necessarilyimply that the neurotransmission of the parasympa-thetic component of the chemoreflex occurs in thissubregion of the NTS. Studies by Finley and Katz (9)have shown dense labeling in the NTSlat after injectionof wheat germ agglutinin-conjugated horseradish per-oxidase into the vascularly isolated carotid body,whereas a functional study by Vardhan et al. (39) hasshown that the NTSmid plays a key role in the neuro-transmission of the sympathoexcitatory component ofthe chemoreflex in the NTS. No study has evaluated theneurotransmission of the parasympathetic componentof the chemoreflex, as we did in the present as well as ina previous investigation from our laboratory (16). Be-cause of this controversy related to the site of termina-tion of the chemoreflex afferents and the processing ofthe sympathetic and parasympathetic components ofthe chemoreflex in different subregions of the NTS, inthe present study we avoided this problem by perform-ing microinjection into the lateral commissural NTS(NTSlat) and into the midline of commissural NTS(NTSmid), or in both (NTSlat1mid), i.e., in all sites inwhich the chemoreflex neurotransmission seems to beprocessed. In this case, we observed that the processingof the parasympathetic component of the chemoreflexseems to be restricted to the NTSlat.

Considering that the antagonism of ionotropic EAAreceptors in the NTSlat or NTSmid with DNQX or Kyn(NTSlat1mid) was not able to block the pressor responseto chemoreflex activation, and also considering previ-ous evidence that metabotropic receptors mediate exci-tatory transmission in the NTS (11–13), we also evalu-ated the possible involvement of this class of EAAreceptors in the processing of the neurotransmission ofthe sympathoexcitatory component of the chemoreflex.The microinjection of MCPG, a metabotropic antago-nist, into the NTSlat or NTSmid also did not modify the



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basal MAP and HR or the pressor and bradycardicresponses to chemoreflex activation, indicating that themetabotropic receptors in the NTS play no major role inthe neurotransmission of the parasympathetic andsympathetic components of the chemoreflex.

The activation of the chemoreflex with KCN inunanesthetized rats produces, in addition to cardiovas-cular and ventilatory responses, an important behav-ioral response (10) which seems to be a consequence ofthe stimulation of the hypothalamic areas involvedwith the defense reaction (25). Considering that chemi-cal or electrical stimulation of these hypothalamicdefense areas increases sympathetic activity (17,30–33), we may suggest that, in contrast to studiesperformed under anesthesia, the pressor response in-duced by chemoreflex activation in the present studydepends at least in part on the hypothalamic defenseareas. It is important to note that the behavioralresponses to KCN, such as exploration of the cage andalertness, were also eliminated by bilateral ligature ofthe carotid body artery (16) or by deafferentation of thecarotid sinus nerve bifurcation (10). Under anesthesia,the alertness and the defense reaction component of thechemoreflex responses are abolished, a fact that mayexplain why in the studies by Vardhan et al. (39) andZhang and Mifflin (40) working with anesthetized rats,the pressor response of the chemoreflex was blocked byEAA receptor antagonists into the NTS. In addition, itis important to note that in the experiments by Vard-han et al. (39) and Zhang and Mifflin (40), no changes inHR were observed in response to chemoreflex activa-tion, whereas in the present study as well as in aprevious study from our laboratory (16) we observed anintense bradycardic response. Therefore, when animalsare under anesthesia, the behavioral and cardiovascu-lar responses to chemoreflex activation are different, asdemonstrated by Franchini and Krieger (10), and thedata obtained in conscious and anesthetized animalscannot be easily compared. The degree of involvementof hypothalamic areas in the generation of the pressorresponse of the chemoreflex in the present study wasnot experimentally addressed, and therefore furtherstudies on unanesthetized rats are required to explorethe possible involvement of hypothalamic projectionsfrom and to the NTS on the processing of the sympatho-excitatory component (pressor response) of the chemo-reflex.

The involvement of other neurotransmitters in theprocessing of the chemoreflex afferents in the NTS hasbeen considered. Studies using microdialysis demon-strated that the release of substance P (22, 35) into theNTS was increased during hypoxia. Adenosine mayalso play a role in the processing of the chemoreflex inthe NTS, especially in the projection from the hypotha-lamic defense areas to the NTS (36, 37). However, therole of these putative neurotransmitters and theirdifferent subtypes of receptors in the processing of thepressor response to the chemoreflex in the lateral andmedial commissural NTS of unanesthetized rats andparticularly in the neurotransmission of the sympatho-

excitatory component of the chemoreflex requires addi-tional studies.

The data of the present study indicate that theneurotransmission of the sympathoexcitatory compo-nent of the chemoreflex is more complex than thecardiovagal component and suggest that neurotransmit-ters other than EAAs may play a key role in theprocessing of the sympathoexcitatory component (pres-sor response) of the chemoreflex in the lateral andmedial commissural NTS.

Perspectives

In a previous study (16), we verified that the brady-cardic response to the chemoreflex activation wasblocked in a dose-dependent manner by AP-5, a selec-tive NMDA receptor antagonist, whereas the pressorresponse was not affected. In accordance with thoseresults, we suggested that the sympathoexcitatorycomponent (pressor response) of the chemoreflex couldbe mediated by non-NMDA receptors at the NTS level,and the experiments presented in the current studywere performed to verify this hypothesis. However, thedata of this study indicated that different ionotropic(Kyn and DNQX) or metabotropic (MCPG) receptorantagonists were not able to block the pressor responseof the chemoreflex, even when Kyn was microinjectedsimultaneously in three different sites of the NTS.Considering that in a previous study an NMDA recep-tor antagonist (AP-5) also produced no blockade of thepressor response of the chemoreflex, we may suggestthat EAA receptors may not be involved in the process-ing of the neurotransmission of the sympathoexcitatorycomponent of the chemoreflex in the NTS. Because ofthis unexpected finding, new and interesting perspec-tives are open to studies involving the neurotransmis-sion of the sympathoexcitatory component of the chemo-reflex in the NTS. One important aspect that must beconsidered in the present study is related to the factthat the activation of the chemoreflex in unanesthe-tized rats, in addition to the cardiovascular and respira-tory changes, produces behavioral responses, whichmay contribute to the pressor response. Therefore,studies involving the possible role of the hypothalamicdefense area, for example, are required to verify ifprojections from this area to the NTS participate in thegeneration of the pressor response to chemoreflex acti-vation. Studies are also required on the possible involve-ment of other areas, including the parabrachial nucleusand periaqueductal gray in the complex physiologicalresponses to the activation of the chemoreflex in unanes-thetized rats, including cardiovascular, respiratory, andbehavioral aspects. In addition to the involvement ofother areas of the brain in this pressor response,another important aspect to be studied, as a conse-quence of the present findings, is related to the possibil-ity that neurotransmitters/neuromodulators other thanEAAs may play a key role in this neurotransmission.Among these potential neurotransmitters/neuromodu-lators of the pressor response of the chemoreflex in theNTS, evidence in the literature indicates the involve-ment of substance P and adenosine in this neurotrans-



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mission. Therefore, experiments using the antagonistof the different subtypes of tachycinergic and puriner-gic receptors in the NTS should be also performed,preferentially in unanesthetized rats, to verify theirpossible involvement in the neurotransmission of thepressor response to chemoreflex activation.

We thank R. F. de Melo for the histological preparations.This work was supported by Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo, Conselho Nacional de Desenvolvimento Cien-tıfico e Tecnologico, and Programa de Apoio aos Nucleos de Excelen-cia.

Address for reprint requests: B. H. Machado, Dept. of Physiology,School of Medicine of Ribeirao Preto, Univ. of Sao Paulo, 14049-900,Ribeirao Preto, Sao Paulo, Brazil.

Received 20 March 1998; accepted in final form 25 August 1998.

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Sympathoexcitatory neurotransmission of the chemoreflex ...€¦ · Sympathoexcitatory neurotransmission of the chemoreflex in the NTS of awake rats ANDREÁ S. HAIBARA,1 LENI G