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Bradycardic and hypotensive responses to microinjection of l -glutamate into the lateral aspect of the commissural NTS are blocked by an NMDA receptor antagonist
Brain Research 852 Ž2000. 68–75 www.elsevier.comrlocaterbres
Research report
Bradycardic and hypotensive responses to microinjection of L-glutamate into the lateral aspect of the commissural NTS are blocked by an NMDA receptor antagonist R.O. Canesin, L.G.H. Bonagamba, B.H. Machado
)
Department of Physiology, School of Medicine of Ribeirao ˜ Preto, UniÕersity of Sao ˜ Paulo, 14049-900, Ribeirao ˜ Preto, SP, Brazil Accepted 5 October 1999
Abstract Baroreflex activation by phenylephrine infusion produces a bradycardic response while microinjection of L-glutamate into the most lateral aspect of the commissural nucleus tractus solitarius ŽNTS, 0.8 mm lateral to the midline. produces bradycardic and hypotensive responses. In the present study we investigated the role of NMDA receptors in the lateral aspect of the commissural NTS Ž0.8 mm lateral to the midline. in the bradycardic and hypotensive responses to microinjection of L-glutamate as well as in the processing of the bradycardic response to the baroreflex activation. The hypotensive and bradycardic responses to L-glutamate microinjection into the NTS were blocked by methyl-atropine Žintravenous, i.v.., indicating that the hypotensive response was secondary to the bradycardia. Microinjection of L-glutamate Ž1 nmolr50 nl. into the NTS was performed before and after microinjection of increasing doses of phosphonovaleric acid ŽAP-5, a selective NMDA antagonist. at the same site. The microinjection of AP-5 w0.5 Ž n s 9., 2.0 Ž n s 8. and 10.0 nmolr50 nl Ž n s 7.x into the NTS Ž0.8 mm lateral to the midline. produced a dose-dependent blockade of the bradycardic and hypotensive responses to L-glutamate. In a specific group of rats the microinjection of 10 nmolr50 nl of AP-5 produced a significant reduction in baroreflex sensitivity 2 min after microinjection into the lateral NTS wgain s y1.48 " 0.12 vs. y0.5 " 0.2 beatsrmmHg, Ž n s 5.x, which was reversible. The data show that the bradycardic responses produced by microinjection of L-glutamate into the most lateral aspect of the commissural NTS or by activation of the baroreflex were blocked by microinjection of AP-5, indicating that the neurotransmission of the parasympathetic component of the baroreflex in the neurons of the lateral aspect of the commissural NTS involves NMDA receptors. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Baroreflex; Excitatory amino acid receptor; Heart rate; Arterial pressure; Parasympathetic tone; AP-5; Methyl-atropine
1. Introduction The nucleus tractus solitarius ŽNTS. is located in the dorsal aspect of the medulla and receives afferent projections from different sensory systems involved in cardiovascular regulation and there is experimental evidence suggesting that the neurotransmission of the baroreflex at the NTS level is mediated by excitatory amino acids ŽEAA. in the NTS w6,17,19,21x. The microinjection of L-glutamate, an excitatory amino acid, into the NTS of anesthetized rats induced hypotension and bradycardia, a typical response of the baroreflex activation w6,9,21x. In a previous study using unanesthetized rats, we showed that microinjection of L-glutamate into the commissural NTS ) Corresponding author. [email protected]
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q 55-16-633-0017;
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0.5 mm lateral to the midline induced an increase in arterial pressure and bradycardia w10x. However, microinjection of L-glutamate into the NTS of the same animals under anesthesia produced a fall in arterial pressure and bradycardia showing that under anesthesia L-glutamate can produce an opposite pattern of arterial pressure response. Several studies from our laboratory w3,4,10–12,14x also documented an increase in arterial pressure and bradycardia in response to the microinjection of L-glutamate into this subregion of the NTS of unanesthetized animals. In preliminary experiments performed in our laboratory, it was also observed that microinjection of L-glutamate into the most lateral aspect of the NTS Ž0.8 mm lateral to midline. in unanesthetized rats produced a fall in arterial pressure and bradycardia. These responses suggested that L-glutamate could be activating both the parasympathoexcitatory and the sympathoinhibitory pathways of the
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 1 9 6 - 4
R.O. Canesin et al.r Brain Research 852 (2000) 68–75
baroreflex. To answer this question we initially evaluated the effect of methyl-atropine Ži.v.. on the bradycardic and hypotensive responses to microinjection of L-glutamate into the lateral aspect of the commissural NTS. The main purpose of the present study was to evaluate the role of NMDA receptors in the lateral aspect of the commissural NTS in Ža. the cardiovascular responses produced by microinjection of L-glutamate and Žb. in the processing of the parasympathetic component of the baroreflex. To achieve these goals we performed the microinjection of AP-5, a selective NMDA receptor antagonist, into the commissural NTS.
2. Materials and methods Male Wistar rats weighing 270–300 g were used in this study. Four days before the experiments, the animals were
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deeply anesthetized with tribromoethanol Ž250 mgrkg i.p.; Aldrich, Milwaukee, WI, USA. and placed in a stereotaxic frame ŽDavid Kopf, Tujunga, CA, USA. for implantation of guide cannulas in the direction of the lateral commissural NTS Ž0.8 mm lateral to the midline. according to the methods described by Michelini and Bonagamba w15x. Guide cannulas of steel Ž17 mm = 0.7 mm. were placed in the holder of the stereotaxic frame. The stereotaxic tower was placed in a vertical position Žzero angulation. and the animal’s head was adjusted with the bregma and lambda at the same level and the antero-posterior, lateral and vertical parameters were determined. The dorso-ventral coordinates for the position of guide cannulas were obtained according to parameters previously used in our laboratory Ž14.5 mm caudal to the bregma and 7.9 mm below the skull. according to the atlas of Paxinos and Watson w18x. With respect to the lateral coordinate, the guide cannulas were introduced 0.8 mm lateral to the midline, in the direction of the
Fig. 1. Typical tracings of one rat representative of the group showing the changes in heart rate ŽHR., pulsatile arterial pressure ŽPAP. and mean arterial pressure ŽMAP. in response to microinjection of L-glutamate Ž1 nmolr50 nl. into the lateral commissural NTS before ŽL-glu control. and 15 min after ŽL-glu 15 min. injection of methyl-atropine Ž2 mgrkg, i.v...
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lateral commissural NTS. One day before the experiments, while rats were under tribromoethanol anesthesia, a catheter ŽPE-10 connected to PE-50; Clay Adams, Parsippany, NJ, USA. was inserted into the abdominal aorta through the femoral artery for measurement of arterial pressure and heart rate. A second catheter was inserted into the femoral vein for intravenous Ži.v.. injection of methyl-atropine or phenylephrine infusion. Both catheters were tunneled subcutaneously and exteriorized through the back of the neck to be connected to the pressure transducer on the subsequent day. Pulsatile arterial pressure ŽPAP. and mean arterial pressure ŽMAP. were measured in conscious freely moving rats with a pressure transducer ŽStatham P23Db. connected to a Narcotrace 80 physiological recorder ŽNarco Bio-Systems, Austin, TX.. Heart rate ŽHR. was measured with a biotachometer ŽNarco Bio System, Austin, TX, USA., activated by signals from the pulsatile arterial pressure channel. Microinjections into the NTS were performed using a 1-ml Hamilton syringe ŽHamilton, Reno, NV, USA. connected by polyethylene PE-10 tubing to an injector needle 1.0 mm longer than the guide cannula in order to reach the lateral commissural NTS. The volume of microinjections was always 50 nl. The cannulas were implanted bilaterally, but the experiments involving the microinjection of Lglutamate and NMDA antagonist were performed unilaterally, while in the experiments involving baroreflex activation the microinjections were performed bilaterally. The time interval between microinjections into the NTS was at least 10 min. Microinjection of L-glutamate into the NTS was performed before and 15 min after injection of methyl-atropine Ži.v... In the experimental protocols related to the blockade of NMDA receptors, we obtained two similar control responses to L-glutamate before the microinjection of AP-5 into the NTS. The microinjections of L-glutamate was repeated at 10 and 60 min after AP-5. The baroreflex was activated by infusion of phenylephrine, an a1 adrenergic agonist, with an infusion pump ŽAti-Orion Sage Model 361.. The venous catheter was connected with polyethylene PE-50 tubing to a 1-ml syringe filled with phenylephrine, 50 mgr1 ml. The infusion of phenylephrine was used to produce an increase of approximately 30 mmHg within a period of 15–20 s and the reflex bradycardia was evaluated at each 10-mmHg increase in MAP. The reflex bradycardia was determined by the HR measured with the biotachometer, which presents an electronic delay of approximately 500 ms. In addition, it is important to note that the time of processing for baroreflex synapses is approximately 700 ms w20x. Therefore, a total delay of 1.2 s was considered when we evaluated the reflex bradycardic response for each corresponding increase of 10 mmHg in MAP. In the experiments related to the baroreflex, the microinjection of AP-5 was performed bilaterally into the NTS and the baroreflex was tested before and 2 and 10 min after bilateral micro-
injection of AP-5 into the lateral aspect of the commissural NTS. The following drugs were used in these experiments: L-glutamate Žexcitatory amino acid., methyl-atropine Žan antagonist of muscarinic receptors., 2-amino-5-phosphonovaleric acid ŽAP-5, a selective antagonist of NMDA receptors. and phenylephrine Žan a1 adrenoceptor agonist.. The results are expressed as mean " S.E.M. For the statistical analysis, the paired Student t-test was used and the level of significance was fixed at p - 0.05. ANOVA was used for comparison of the effect of three different doses of AP-5 on the cardiovascular responses to micro-
Fig. 2. Changes in heart rate Ž DHR. wupper panelx and in mean arterial pressure Ž DMAP. wbottom panel Bx in response to microinjection of saline Žcontrol. and L-glutamate into the lateral commissural NTS before ŽL-glu control. and after intravenous injection of methyl-atropine ŽL-glu after atropine.. U p- 0.05, compared to L-glu control; q p- 0.05, compared to saline.
R.O. Canesin et al.r Brain Research 852 (2000) 68–75
injection of L-glutamate. The data related to the gain of the baroreflex before and after bilateral microinjection of AP-5 into the most lateral aspect of the NTS were submitted to one way ANOVA for multiple comparison to verify the statistical differences Ž p - 0.05., followed by a Tukey post-test when appropriate.
3. Results Fig. 1 is a typical tracing of one rat representative of the group showing the hypotensive and bradycardic response to microinjection of L-glutamate into the lateral aspect of the commissural NTS before and 15 min after methyl-atropine Ži.v... Fig. 2 summarizes the data and shows that microinjection of L-glutamate into the lateral aspect of the commissural NTS produced bradycardia Župper panel, y100 " 21 vs. q4 " 7 bpm. and hypotension Žbottom panel, y35 " 5 vs. q13 " 7 mmHg., which were completely blocked by methyl-atropine, indicating that the excitation of the parasympathetic component is the major cardiovascular response induced by the activation of this specific site of the NTS and that the hypotensive response is secondary to this intense bradycardia. Note that after
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methyl-atropine a tendency to an increase in MAP and HR was observed. Fig. 3 shows tracings of three rats representative of their respective group Žpanels A, B and C.. The tracings show that the bradycardia and hypotensive responses to microinjection of L-glutamate into the commissural lateral NTS were blocked in a dose-dependent manner by microinjection of 0.5, 2 and 10 nmolr50 nl of AP-5. Fig. 4 summarizes the data and shows that the microinjection of increasing doses of AP-5 produced a dose-dependent blockade of the bradycardic Žy138 " 25; y59 " 18 and y10 " 9 bpm. and hypotensive responses Žy36 " 8; y15 " 5 and y3 " 3 mmHg. to L-glutamate microinjection. Fig. 5 shows the linear regression lines of the gain of the baroreflex before and 2 and 10 min after bilateral microinjection of AP-5 into the most lateral aspect of the commissural NTS. When we performed baroreflex activation by phenylephrine infusion before and after bilateral microinjection of AP-5 into the lateral commissural NTS, we observed that the gain of reflex was significantly reduced 2 min after microinjection of AP-5, showing that NMDA receptors in this most lateral subregion of the commissural NTS play a key role in the neurotransmission of the baroreflex. Note that 10 min after microinjection of AP-5 the gain of the baroreflex was back to the control level.
Fig. 3. Typical tracings of three rats representative of their respective groups showing the changes in heart rate ŽHR., pulsatile arterial pressure ŽPAP. and mean arterial pressure ŽMAP. in response to L-glutamate Ž1 nmolr50 nl. microinjected before and 10 min after microinjection of increasing doses of AP-5 w0.5 ŽA.; 2.0 ŽB. and 10 nmolr50 nl ŽC.x into the lateral commissural NTS.
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Fig. 4. Changes in heart rate Ž DHR. wupper panelx and mean arterial pressure Ž DMAP. wbottom panelx in response to the microinjection of L-glutamate Ž1 nmolr50 nl. into the NTS of three different groups of animals before and 10 and 60 min after the microinjection of three different doses of AP-5 into the lateral commissural NTS Ž0.5, 2.0 and 10 nmolr50 nl.. U p- 0.05, compared to the respective control.
the midline. of anesthetized animals w21x. Studies by Machado and Bonagamba w10x showed that microinjection of L-glutamate into this subregion of the NTS, in unanesthetized rats, produced an increase in arterial pressure and bradycardia. Therefore, the results of the present study show that the microinjection of L-glutamate into the most lateral aspect of the NTS, encompassing the tractus solitarius, presented a hypotensive response, which is the opposite in relation to the pressor response to microinjection of the same dose of L-glutamate performed 0.3 mm more medial in the commissural NTS. A study by Nelson et al. performed on anesthetized rats w16x documented that microinjection of L-glutamate into the caudal NTS produced an increase in arterial pressure, while microinjection into the lateral commissural NTS produced a fall in arterial pressure, indicating that subregions of the NTS contain a heterogeneity of neurons involved in different aspects of the autonomic regulation of cardiovascular function. Our results also show that the subregion 0.8 mm lateral to the midline seems to be preferentially involved in the processing of the bradycardic response because methyl-atropine, a muscarinic antagonist, also blocked the hypotension, indicating that the fall in arterial pressure was secondary to the intense reduction in heart rate and consequently in cardiac output. Previous studies from our laboratory w2,4,7x showed that the parasympathetic component of the chemoreflex and Bezold–Jarish reflex, as well as in response to microinjection of L-glutamate into the NTS Ž0.5 mm lateral to the midline. seems to be mediated by NMDA receptors because the bradycardic response was blocked in a dose-dependent manner by AP-5 microinjected into the NTS. In relation to the baroreflex, a study by Tian and Hartle w22x performed on anesthetized rats documented that infusion of MK-801, a noncompetitive NMDA receptor antagonist, into the NTS attenuated the reflex bradycardia in response
Fig. 6 is a photomicrograph of a transversal section of the brainstem of two rats representative of their respective groups showing the site where the microinjection was performed in the most lateral aspect of the commissural NTS Ž0.8 mm lateral to the midline. unilaterally Župper panel. or bilaterally Žbottom panel..
4. Discussion The microinjection of L-glutamate into the most lateral aspect of the commissural NTS Ž0.8 mm lateral to midline. produced bradycardic and hypotensive responses in unanesthetized rats. Previous studies have documented that this response pattern is observed when L-glutamate is microinjected into the commissural NTS Ž0.5 mm lateral to
Fig. 5. Linear regressions of the gain of the baroreflex in response to an increase in arterial pressure induced by the infusion of phenylephrine Ži.v.. before and 2 and 10 min after bilateral microinjection of AP-5 Ž10 nmolr50 nl. into the lateral commissural NTS Ž ns 5.. U p- 0.05, compared to control.
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Fig. 6. Photomicrographs of transverse sections of the brain stem showing unilateral Žpanel A. and bilateral Žpanel B. microinjection sites in the lateral commissural NTS. Arrows indicate the sites of microinjections in the NTS marked with Evan’s blue dye.
to phenylephrine injection. In studies performed in unanesthetized rats, we observed that bilateral microinjection of AP-5 into the commissural NTS Ž0.5 mm lateral to the midline. produced a significant reduction in the gain of the baroreflex, indicating an important role for NMDA receptors in the processing of the parasympathetic component of the baroreflex in this subregion of the NTS w13x. In the present study we also verified that the bradycardic and hypotensive responses to L-glutamate were blocked in a dose-dependent manner by AP-5, a selective NMDA receptor antagonist. Considering that methyl-atropine also blocked the bradycardia and hypotension occur-
ring in response to L-glutamate, we may suggest that AP-5 blocked NMDA receptors related to the processing of the bradycardic component of the response to L-glutamate and consequently the hypotensive response. We have indirect evidence that the sympatho-inhibitory component, at least in the commissural NTS Ž0.5 mm lateral to the midline., is mediated by non-NMDA receptors because microinjection of DNQX, a non-NMDA selective antagonist, at this level produced a marked increase in baseline MAP w8x, while microinjection of AP-5 at the same site of the NTS w2,4,7x as well as in the more lateral aspect in the present study produced no changes in baseline MAP.
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Studies by Donoghue et al. w5x using an electrophysiological approach in cats documented that the baroreceptor and chemoreceptor afferents project to different subregions of the NTS, and that the baroreceptors send their projections to the most lateral portions of the rostral NTS and ipsilateral portions of the commissural subnucleus, suggesting that the most lateral aspect of the NTS is integral to baroreflex pathways. Studies by Ciriello et al. w1x in rats documented that the first synapse of the baroreceptor afferents is located at the most lateral aspect of the commissural NTS. In the present study we obtained additional evidence in favor of the possibility that the most lateral aspect of the commissural NTS in rats is part of the baroreflex pathways, considering that bilateral microinjection of AP-5 into this subregion produced a significant reduction in the bradycardic response to baroreflex activation. In these experiments our initial aim was to study the sympathoinhibitory component of the baroreflex because microinjections of L-glutamate in this subregion of the NTS Ž0.8 mm lateral to midline. produced hypotension in addition to the bradycardic response. However, the data show that the fall in arterial pressure was not due to sympathoinhibition because methyl-atropine blocked both the bradycardic and hypotensive responses to L-glutamate microinjection. In previous studies, we showed that the parasympathetic component of the baroreflex was blocked by bilateral microinjection of kynurenic acid, a nonselective antagonist of EAA receptors, or by AP-5 microinjected into the commissural NTS 0.5 mm lateral to the midline w3,12,13x. Therefore, we have evidence that the parasympathetic component of the baroreflex, chemoreflex w7x and Bezold–Jarisch reflex w2x in the commissural NTS is mediated by NMDA receptors. The present study was performed in a region 0.3 mm more lateral than the subregion explored in the previous studies from our laboratory w7,8,10–14x and the findings clearly indicate that NMDA receptors located in the cell bodies of this subregion, probably in the tractus solitarius, also play a key role in the processing of the parasympathetic component of the baroreflex. We conclude that the most lateral aspect of the commissural NTS seems to be preferentially involved in the neurotransmission of the parasympathetic component of the baroreflex and the evidence indicates that this neurotransmission is processed by NMDA receptors.
Acknowledgements We thank Rubens F. Melo for the histological preparations. This work was supported by Fundac¸ao ˜ de Amparo a` Pesquisa do Estado de Sao ˜ Paulo ŽFAPESP., Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico ´ ´ ŽCNPQ. and Programa Nacional de Apoio aos Nucleos de ´ ŽPRONEX.. Excelencia ˆ
References w1x J. Ciriello, S.L. Hochstenbach, S. Roder, Central projections of baroreceptor and chemoreceptor afferent fibers in the rat, in: I. Robin A. Barraco ŽEd.., Nucleus of the Solitary Tract, CRC Press, Boca Raton, 1994. w2x D.A. Chianca Jr., B.H. Machado, Microinjection of NMDA antagonist into the NTS of conscious rats blocks the Bezold–Jarish reflex, Brain Res. 718 Ž1996. 185–188. w3x E. Colombari, L.G.H. Bonagamba, B.H. Machado, Mechanisms of pressor and bradycardic responses to L-glutamate microinjected into the NTS of conscious rats, Am. J. Physiol. 266 Ž1994. R730–R738, Regul. Integr. Comp. Physiol., 35. w4x E. Colombari, L.G.H. Bonagamba, B.H. Machado, NMDA receptor antagonist blocks the bradycardic but not the pressor response to L-glutamate microinjected into the NTS of unanesthetized rats, Brain Res. 749 Ž1997. 209–213. w5x S. Donoghue, R.B. Felder, D. Jordan, K.M. Spyer, The central projections of carotid baroreceptors and chemoreceptors in the cat: a neurophysiological study, J. Physiol. 347 Ž1984. 397–409. w6x F.J. Gordon, W.T. Talman, Role of excitatory amino acids and their receptors in bulbospinal control of cardiovascular function, in: G. Kunos, J. Ciriello ŽEds.., Central Neural Mechanisms in Cardiovascular Regulation, Vol. 2, Birkauser, Boston, MA, 1992, pp. 209–225. w7x A.S. Haibara, E. Colombari, D.A. Chianca Jr., L.G.H. Bonagamba, B.H. Machado, NMDA receptors in NTS are involved in bradycardic but not in pressor response of chemoreflex, Am. J. Physiol. 269 Ž1995. H1421–H1427, Heart Circ. Physiol., 38. w8x A.S. Haibara, L.G.H. Bonagamba, B.H. Machado, Sympathoexcitatory neurotransmission of chemoreflex in the NTS of awake rats, Am. J. Physiol. 276 Ž1999. R69–R80, Regul. Integr. Comp. Physiol., 45. w9x C. Leone, F.J. Gordon, Is L-glutamate a neurotransmitter of baroreceptor information in the nucleus of the tractus solitarius?, J. Pharmacol. Exp. Ther. 250 Ž1989. 953–962. w10x B.H. Machado, L.G.H. Bonagamba, Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats, Brain Res. 576 Ž1992. 131–138. w11x B.H. Machado, L.G.H. Bonagamba, Microinjection of S-nitrosocysteine into the nucleus tractus solitarii of conscious rats decreases arterial pressure but L-glutamate does not, Eur. J. Pharmacol. 221 Ž1992. 179–182. w12x B.H. Machado, H. Mauad, D.A. Chianca Jr., A.S. Haibara, E. Colombari, Autonomic processing of the cardiovascular reflexes in the nucleus tractus solitarii, Braz. J. Med. Biol. Res. 30 Ž1997. 533–543. w13x B.H. Machado, M. Frigero, L.G.H. Bonagamba, R. Okamoto, NMDA receptors in the NTS mediate bradycardic responses to L-glutamate microinjection or to baroreflex activation, Soc. Neurosci. Abstr. 23 Ž1997. 656, Part 1. w14x H. Mauad, B.H. Machado, Glycine blocks the pressor response to L-glutamate microinjected into the nucleus tractus solitarii of conscious rats, Braz. J. Med. Biol. Res. 28 Ž1995. 699–704. w15x L.C. Michelini, L.G.H. Bonagamba, Baroreceptor reflex modulation by vasopressin microinjected into the nucleus tractus solitarii of conscious rats, Hypertension 11 Ž1988. 75–79. w16x D.O. Nelson, H.L. Cohen, J.L. Feldman, D.R. McCrimmon, Cardiovascular function is altered by picomole injections of glutamate into rat medulla, J. Neurosci. 8 Ž1988. 1684–1693. w17x H. Ohta, W.T. Talman, Both NMDA and non-NMDA receptors in the NTS participate in the baroreceptor reflex in rats, Am. J. Physiol. 267 Ž1997. R1065–R1070. w18x G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. w19x K.M. Spyer, The central nervous organization of reflex circulatory control, in: A.D. Loewy, K.M. Spyer ŽEds.., Central Regulation of Autonomic Functions, Oxford Univ. Press, New York, 1990.
R.O. Canesin et al.r Brain Research 852 (2000) 68–75 w20x D.F. Su, C. Cerruti, C. Barres, C. Julien, M. Vincent, C. Paultre, J. Sassard, Arterial baroreflex control of heart rate period is not related to blood pressure variability in conscious hypertensive and normotensive rats, Clin. Exp. Pharmacol. Physiol. 19 Ž1992. 767–775. w21x W.T. Talman, M.H. Perrone, D.J. Reis, Evidence of L-glutamate as
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the neurotransmitter of baroreceptor afferent nerve fibers, Science 209 Ž1980. 813–815. w22x B. Tian, D.K. Hartle, Cardiovascular effects of NMDA and MK-801 infusion at area postrema and mNTS in rat, Pharmacol., Biochem. Behavior 49 Ž1994. 489–495.
Research report
Bradycardic and hypotensive responses to microinjection of L-glutamate into the lateral aspect of the commissural NTS are blocked by an NMDA receptor antagonist R.O. Canesin, L.G.H. Bonagamba, B.H. Machado
)
Department of Physiology, School of Medicine of Ribeirao ˜ Preto, UniÕersity of Sao ˜ Paulo, 14049-900, Ribeirao ˜ Preto, SP, Brazil Accepted 5 October 1999
Abstract Baroreflex activation by phenylephrine infusion produces a bradycardic response while microinjection of L-glutamate into the most lateral aspect of the commissural nucleus tractus solitarius ŽNTS, 0.8 mm lateral to the midline. produces bradycardic and hypotensive responses. In the present study we investigated the role of NMDA receptors in the lateral aspect of the commissural NTS Ž0.8 mm lateral to the midline. in the bradycardic and hypotensive responses to microinjection of L-glutamate as well as in the processing of the bradycardic response to the baroreflex activation. The hypotensive and bradycardic responses to L-glutamate microinjection into the NTS were blocked by methyl-atropine Žintravenous, i.v.., indicating that the hypotensive response was secondary to the bradycardia. Microinjection of L-glutamate Ž1 nmolr50 nl. into the NTS was performed before and after microinjection of increasing doses of phosphonovaleric acid ŽAP-5, a selective NMDA antagonist. at the same site. The microinjection of AP-5 w0.5 Ž n s 9., 2.0 Ž n s 8. and 10.0 nmolr50 nl Ž n s 7.x into the NTS Ž0.8 mm lateral to the midline. produced a dose-dependent blockade of the bradycardic and hypotensive responses to L-glutamate. In a specific group of rats the microinjection of 10 nmolr50 nl of AP-5 produced a significant reduction in baroreflex sensitivity 2 min after microinjection into the lateral NTS wgain s y1.48 " 0.12 vs. y0.5 " 0.2 beatsrmmHg, Ž n s 5.x, which was reversible. The data show that the bradycardic responses produced by microinjection of L-glutamate into the most lateral aspect of the commissural NTS or by activation of the baroreflex were blocked by microinjection of AP-5, indicating that the neurotransmission of the parasympathetic component of the baroreflex in the neurons of the lateral aspect of the commissural NTS involves NMDA receptors. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Baroreflex; Excitatory amino acid receptor; Heart rate; Arterial pressure; Parasympathetic tone; AP-5; Methyl-atropine
1. Introduction The nucleus tractus solitarius ŽNTS. is located in the dorsal aspect of the medulla and receives afferent projections from different sensory systems involved in cardiovascular regulation and there is experimental evidence suggesting that the neurotransmission of the baroreflex at the NTS level is mediated by excitatory amino acids ŽEAA. in the NTS w6,17,19,21x. The microinjection of L-glutamate, an excitatory amino acid, into the NTS of anesthetized rats induced hypotension and bradycardia, a typical response of the baroreflex activation w6,9,21x. In a previous study using unanesthetized rats, we showed that microinjection of L-glutamate into the commissural NTS ) Corresponding author. [email protected]
Fax:
q 55-16-633-0017;
e-mail:
0.5 mm lateral to the midline induced an increase in arterial pressure and bradycardia w10x. However, microinjection of L-glutamate into the NTS of the same animals under anesthesia produced a fall in arterial pressure and bradycardia showing that under anesthesia L-glutamate can produce an opposite pattern of arterial pressure response. Several studies from our laboratory w3,4,10–12,14x also documented an increase in arterial pressure and bradycardia in response to the microinjection of L-glutamate into this subregion of the NTS of unanesthetized animals. In preliminary experiments performed in our laboratory, it was also observed that microinjection of L-glutamate into the most lateral aspect of the NTS Ž0.8 mm lateral to midline. in unanesthetized rats produced a fall in arterial pressure and bradycardia. These responses suggested that L-glutamate could be activating both the parasympathoexcitatory and the sympathoinhibitory pathways of the
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 1 9 6 - 4
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baroreflex. To answer this question we initially evaluated the effect of methyl-atropine Ži.v.. on the bradycardic and hypotensive responses to microinjection of L-glutamate into the lateral aspect of the commissural NTS. The main purpose of the present study was to evaluate the role of NMDA receptors in the lateral aspect of the commissural NTS in Ža. the cardiovascular responses produced by microinjection of L-glutamate and Žb. in the processing of the parasympathetic component of the baroreflex. To achieve these goals we performed the microinjection of AP-5, a selective NMDA receptor antagonist, into the commissural NTS.
2. Materials and methods Male Wistar rats weighing 270–300 g were used in this study. Four days before the experiments, the animals were
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deeply anesthetized with tribromoethanol Ž250 mgrkg i.p.; Aldrich, Milwaukee, WI, USA. and placed in a stereotaxic frame ŽDavid Kopf, Tujunga, CA, USA. for implantation of guide cannulas in the direction of the lateral commissural NTS Ž0.8 mm lateral to the midline. according to the methods described by Michelini and Bonagamba w15x. Guide cannulas of steel Ž17 mm = 0.7 mm. were placed in the holder of the stereotaxic frame. The stereotaxic tower was placed in a vertical position Žzero angulation. and the animal’s head was adjusted with the bregma and lambda at the same level and the antero-posterior, lateral and vertical parameters were determined. The dorso-ventral coordinates for the position of guide cannulas were obtained according to parameters previously used in our laboratory Ž14.5 mm caudal to the bregma and 7.9 mm below the skull. according to the atlas of Paxinos and Watson w18x. With respect to the lateral coordinate, the guide cannulas were introduced 0.8 mm lateral to the midline, in the direction of the
Fig. 1. Typical tracings of one rat representative of the group showing the changes in heart rate ŽHR., pulsatile arterial pressure ŽPAP. and mean arterial pressure ŽMAP. in response to microinjection of L-glutamate Ž1 nmolr50 nl. into the lateral commissural NTS before ŽL-glu control. and 15 min after ŽL-glu 15 min. injection of methyl-atropine Ž2 mgrkg, i.v...
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lateral commissural NTS. One day before the experiments, while rats were under tribromoethanol anesthesia, a catheter ŽPE-10 connected to PE-50; Clay Adams, Parsippany, NJ, USA. was inserted into the abdominal aorta through the femoral artery for measurement of arterial pressure and heart rate. A second catheter was inserted into the femoral vein for intravenous Ži.v.. injection of methyl-atropine or phenylephrine infusion. Both catheters were tunneled subcutaneously and exteriorized through the back of the neck to be connected to the pressure transducer on the subsequent day. Pulsatile arterial pressure ŽPAP. and mean arterial pressure ŽMAP. were measured in conscious freely moving rats with a pressure transducer ŽStatham P23Db. connected to a Narcotrace 80 physiological recorder ŽNarco Bio-Systems, Austin, TX.. Heart rate ŽHR. was measured with a biotachometer ŽNarco Bio System, Austin, TX, USA., activated by signals from the pulsatile arterial pressure channel. Microinjections into the NTS were performed using a 1-ml Hamilton syringe ŽHamilton, Reno, NV, USA. connected by polyethylene PE-10 tubing to an injector needle 1.0 mm longer than the guide cannula in order to reach the lateral commissural NTS. The volume of microinjections was always 50 nl. The cannulas were implanted bilaterally, but the experiments involving the microinjection of Lglutamate and NMDA antagonist were performed unilaterally, while in the experiments involving baroreflex activation the microinjections were performed bilaterally. The time interval between microinjections into the NTS was at least 10 min. Microinjection of L-glutamate into the NTS was performed before and 15 min after injection of methyl-atropine Ži.v... In the experimental protocols related to the blockade of NMDA receptors, we obtained two similar control responses to L-glutamate before the microinjection of AP-5 into the NTS. The microinjections of L-glutamate was repeated at 10 and 60 min after AP-5. The baroreflex was activated by infusion of phenylephrine, an a1 adrenergic agonist, with an infusion pump ŽAti-Orion Sage Model 361.. The venous catheter was connected with polyethylene PE-50 tubing to a 1-ml syringe filled with phenylephrine, 50 mgr1 ml. The infusion of phenylephrine was used to produce an increase of approximately 30 mmHg within a period of 15–20 s and the reflex bradycardia was evaluated at each 10-mmHg increase in MAP. The reflex bradycardia was determined by the HR measured with the biotachometer, which presents an electronic delay of approximately 500 ms. In addition, it is important to note that the time of processing for baroreflex synapses is approximately 700 ms w20x. Therefore, a total delay of 1.2 s was considered when we evaluated the reflex bradycardic response for each corresponding increase of 10 mmHg in MAP. In the experiments related to the baroreflex, the microinjection of AP-5 was performed bilaterally into the NTS and the baroreflex was tested before and 2 and 10 min after bilateral micro-
injection of AP-5 into the lateral aspect of the commissural NTS. The following drugs were used in these experiments: L-glutamate Žexcitatory amino acid., methyl-atropine Žan antagonist of muscarinic receptors., 2-amino-5-phosphonovaleric acid ŽAP-5, a selective antagonist of NMDA receptors. and phenylephrine Žan a1 adrenoceptor agonist.. The results are expressed as mean " S.E.M. For the statistical analysis, the paired Student t-test was used and the level of significance was fixed at p - 0.05. ANOVA was used for comparison of the effect of three different doses of AP-5 on the cardiovascular responses to micro-
Fig. 2. Changes in heart rate Ž DHR. wupper panelx and in mean arterial pressure Ž DMAP. wbottom panel Bx in response to microinjection of saline Žcontrol. and L-glutamate into the lateral commissural NTS before ŽL-glu control. and after intravenous injection of methyl-atropine ŽL-glu after atropine.. U p- 0.05, compared to L-glu control; q p- 0.05, compared to saline.
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injection of L-glutamate. The data related to the gain of the baroreflex before and after bilateral microinjection of AP-5 into the most lateral aspect of the NTS were submitted to one way ANOVA for multiple comparison to verify the statistical differences Ž p - 0.05., followed by a Tukey post-test when appropriate.
3. Results Fig. 1 is a typical tracing of one rat representative of the group showing the hypotensive and bradycardic response to microinjection of L-glutamate into the lateral aspect of the commissural NTS before and 15 min after methyl-atropine Ži.v... Fig. 2 summarizes the data and shows that microinjection of L-glutamate into the lateral aspect of the commissural NTS produced bradycardia Župper panel, y100 " 21 vs. q4 " 7 bpm. and hypotension Žbottom panel, y35 " 5 vs. q13 " 7 mmHg., which were completely blocked by methyl-atropine, indicating that the excitation of the parasympathetic component is the major cardiovascular response induced by the activation of this specific site of the NTS and that the hypotensive response is secondary to this intense bradycardia. Note that after
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methyl-atropine a tendency to an increase in MAP and HR was observed. Fig. 3 shows tracings of three rats representative of their respective group Žpanels A, B and C.. The tracings show that the bradycardia and hypotensive responses to microinjection of L-glutamate into the commissural lateral NTS were blocked in a dose-dependent manner by microinjection of 0.5, 2 and 10 nmolr50 nl of AP-5. Fig. 4 summarizes the data and shows that the microinjection of increasing doses of AP-5 produced a dose-dependent blockade of the bradycardic Žy138 " 25; y59 " 18 and y10 " 9 bpm. and hypotensive responses Žy36 " 8; y15 " 5 and y3 " 3 mmHg. to L-glutamate microinjection. Fig. 5 shows the linear regression lines of the gain of the baroreflex before and 2 and 10 min after bilateral microinjection of AP-5 into the most lateral aspect of the commissural NTS. When we performed baroreflex activation by phenylephrine infusion before and after bilateral microinjection of AP-5 into the lateral commissural NTS, we observed that the gain of reflex was significantly reduced 2 min after microinjection of AP-5, showing that NMDA receptors in this most lateral subregion of the commissural NTS play a key role in the neurotransmission of the baroreflex. Note that 10 min after microinjection of AP-5 the gain of the baroreflex was back to the control level.
Fig. 3. Typical tracings of three rats representative of their respective groups showing the changes in heart rate ŽHR., pulsatile arterial pressure ŽPAP. and mean arterial pressure ŽMAP. in response to L-glutamate Ž1 nmolr50 nl. microinjected before and 10 min after microinjection of increasing doses of AP-5 w0.5 ŽA.; 2.0 ŽB. and 10 nmolr50 nl ŽC.x into the lateral commissural NTS.
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Fig. 4. Changes in heart rate Ž DHR. wupper panelx and mean arterial pressure Ž DMAP. wbottom panelx in response to the microinjection of L-glutamate Ž1 nmolr50 nl. into the NTS of three different groups of animals before and 10 and 60 min after the microinjection of three different doses of AP-5 into the lateral commissural NTS Ž0.5, 2.0 and 10 nmolr50 nl.. U p- 0.05, compared to the respective control.
the midline. of anesthetized animals w21x. Studies by Machado and Bonagamba w10x showed that microinjection of L-glutamate into this subregion of the NTS, in unanesthetized rats, produced an increase in arterial pressure and bradycardia. Therefore, the results of the present study show that the microinjection of L-glutamate into the most lateral aspect of the NTS, encompassing the tractus solitarius, presented a hypotensive response, which is the opposite in relation to the pressor response to microinjection of the same dose of L-glutamate performed 0.3 mm more medial in the commissural NTS. A study by Nelson et al. performed on anesthetized rats w16x documented that microinjection of L-glutamate into the caudal NTS produced an increase in arterial pressure, while microinjection into the lateral commissural NTS produced a fall in arterial pressure, indicating that subregions of the NTS contain a heterogeneity of neurons involved in different aspects of the autonomic regulation of cardiovascular function. Our results also show that the subregion 0.8 mm lateral to the midline seems to be preferentially involved in the processing of the bradycardic response because methyl-atropine, a muscarinic antagonist, also blocked the hypotension, indicating that the fall in arterial pressure was secondary to the intense reduction in heart rate and consequently in cardiac output. Previous studies from our laboratory w2,4,7x showed that the parasympathetic component of the chemoreflex and Bezold–Jarish reflex, as well as in response to microinjection of L-glutamate into the NTS Ž0.5 mm lateral to the midline. seems to be mediated by NMDA receptors because the bradycardic response was blocked in a dose-dependent manner by AP-5 microinjected into the NTS. In relation to the baroreflex, a study by Tian and Hartle w22x performed on anesthetized rats documented that infusion of MK-801, a noncompetitive NMDA receptor antagonist, into the NTS attenuated the reflex bradycardia in response
Fig. 6 is a photomicrograph of a transversal section of the brainstem of two rats representative of their respective groups showing the site where the microinjection was performed in the most lateral aspect of the commissural NTS Ž0.8 mm lateral to the midline. unilaterally Župper panel. or bilaterally Žbottom panel..
4. Discussion The microinjection of L-glutamate into the most lateral aspect of the commissural NTS Ž0.8 mm lateral to midline. produced bradycardic and hypotensive responses in unanesthetized rats. Previous studies have documented that this response pattern is observed when L-glutamate is microinjected into the commissural NTS Ž0.5 mm lateral to
Fig. 5. Linear regressions of the gain of the baroreflex in response to an increase in arterial pressure induced by the infusion of phenylephrine Ži.v.. before and 2 and 10 min after bilateral microinjection of AP-5 Ž10 nmolr50 nl. into the lateral commissural NTS Ž ns 5.. U p- 0.05, compared to control.
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Fig. 6. Photomicrographs of transverse sections of the brain stem showing unilateral Žpanel A. and bilateral Žpanel B. microinjection sites in the lateral commissural NTS. Arrows indicate the sites of microinjections in the NTS marked with Evan’s blue dye.
to phenylephrine injection. In studies performed in unanesthetized rats, we observed that bilateral microinjection of AP-5 into the commissural NTS Ž0.5 mm lateral to the midline. produced a significant reduction in the gain of the baroreflex, indicating an important role for NMDA receptors in the processing of the parasympathetic component of the baroreflex in this subregion of the NTS w13x. In the present study we also verified that the bradycardic and hypotensive responses to L-glutamate were blocked in a dose-dependent manner by AP-5, a selective NMDA receptor antagonist. Considering that methyl-atropine also blocked the bradycardia and hypotension occur-
ring in response to L-glutamate, we may suggest that AP-5 blocked NMDA receptors related to the processing of the bradycardic component of the response to L-glutamate and consequently the hypotensive response. We have indirect evidence that the sympatho-inhibitory component, at least in the commissural NTS Ž0.5 mm lateral to the midline., is mediated by non-NMDA receptors because microinjection of DNQX, a non-NMDA selective antagonist, at this level produced a marked increase in baseline MAP w8x, while microinjection of AP-5 at the same site of the NTS w2,4,7x as well as in the more lateral aspect in the present study produced no changes in baseline MAP.
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Studies by Donoghue et al. w5x using an electrophysiological approach in cats documented that the baroreceptor and chemoreceptor afferents project to different subregions of the NTS, and that the baroreceptors send their projections to the most lateral portions of the rostral NTS and ipsilateral portions of the commissural subnucleus, suggesting that the most lateral aspect of the NTS is integral to baroreflex pathways. Studies by Ciriello et al. w1x in rats documented that the first synapse of the baroreceptor afferents is located at the most lateral aspect of the commissural NTS. In the present study we obtained additional evidence in favor of the possibility that the most lateral aspect of the commissural NTS in rats is part of the baroreflex pathways, considering that bilateral microinjection of AP-5 into this subregion produced a significant reduction in the bradycardic response to baroreflex activation. In these experiments our initial aim was to study the sympathoinhibitory component of the baroreflex because microinjections of L-glutamate in this subregion of the NTS Ž0.8 mm lateral to midline. produced hypotension in addition to the bradycardic response. However, the data show that the fall in arterial pressure was not due to sympathoinhibition because methyl-atropine blocked both the bradycardic and hypotensive responses to L-glutamate microinjection. In previous studies, we showed that the parasympathetic component of the baroreflex was blocked by bilateral microinjection of kynurenic acid, a nonselective antagonist of EAA receptors, or by AP-5 microinjected into the commissural NTS 0.5 mm lateral to the midline w3,12,13x. Therefore, we have evidence that the parasympathetic component of the baroreflex, chemoreflex w7x and Bezold–Jarisch reflex w2x in the commissural NTS is mediated by NMDA receptors. The present study was performed in a region 0.3 mm more lateral than the subregion explored in the previous studies from our laboratory w7,8,10–14x and the findings clearly indicate that NMDA receptors located in the cell bodies of this subregion, probably in the tractus solitarius, also play a key role in the processing of the parasympathetic component of the baroreflex. We conclude that the most lateral aspect of the commissural NTS seems to be preferentially involved in the neurotransmission of the parasympathetic component of the baroreflex and the evidence indicates that this neurotransmission is processed by NMDA receptors.
Acknowledgements We thank Rubens F. Melo for the histological preparations. This work was supported by Fundac¸ao ˜ de Amparo a` Pesquisa do Estado de Sao ˜ Paulo ŽFAPESP., Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico ´ ´ ŽCNPQ. and Programa Nacional de Apoio aos Nucleos de ´ ŽPRONEX.. Excelencia ˆ
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