- Email: [email protected]
Involvement of the ipsilateral rostral ventrolateral medulla in the pressor response to l -glutamate microinjection into the nucleus tractus solitarii of awake rats
Journal of the Autonomic Nervous System 74 Ž1998. 43–48
Involvement of the ipsilateral rostral ventrolateral medulla in the pressor response to L-glutamate microinjection into the nucleus tractus solitarii of awake rats Helder Mauad b, Benedito H. Machado ´ a
a,)
Department of Physiology, School of Medicine of Ribeirao ˜ Preto, UniÕersity of Sao ˜ Paulo, 14049-900, Ribeirao ˜ Preto, SP, Brazil Department of Physiological Sciences, Biomedical Center, Federal UniÕersity of Espirito Santo, 29040-090, Vitoria, ´ ES, Brazil
b
Received 15 April 1998; accepted 27 July 1998
Abstract Microinjection of L-glutamate into the lateral commissural nucleus tractus solitarii ŽNTS. of unanesthetized rats evokes increases in mean arterial pressure ŽMAP. and a bradycardia. In a previous study we verified that this increase in MAP is mediated sympathetically because prazosin Živ. blocks this response. The aim of the present study was to evaluate the role of the rostral ventrolateral medulla ŽRVLM. in the pressor response produced by L-glutamate microinjected into the NTS of unanesthetized rats. L-Glutamate was microinjected into the NTS before and 15 and 90 min after microinjection of kynurenic acid into the ipsilateral RVLM. Pressor Žq24 " 3 vs. q6 " 3 mm Hg. and bradycardic Žy101" 10 vs. y3 " 12 bpm. responses to L-glutamate microinjected into the NTS Ž n s 8. were almost abolished 15 min after microinjection of kynurenic acid into the RVLM when compared with control responses. Both pressor Žq23 " 6 mm Hg. and bradycardic Žy93 " 16 bpm. responses to L-glutamate into the NTS returned to control values 90 min after microinjection of kynurenic acid into the RVLM. These data indicate that the pressor response to L-glutamate into the NTS is essentially dependent on the ipsilateral RVLM and also that this sympatho-excitatory response is mediated by excitatory amino acid receptors in RVLM neurons. q 1998 Elsevier Science B.V. All rights reserved. Keywords: EAA receptors; Chemoreflex; Cardiovascular regulation; Ionotropic receptors; Sympatho-excitation; Neurotransmitter
1. Introduction Several studies have indicated that the excitatory amino acid L-glutamate seems to play a key role in the neurotransmission of the baro- ŽTalman et al., 1980; Gordon and Talman, 1992; Machado and Bonagamba, 1992; Colombari et al., 1994., chemo- ŽVardhan et al., 1993; Zhang and Mifflin, 1993; Haibara et al., 1995. and Bezold–Jarisch reflex ŽChianca and Machado, 1996. in the nucleus tractus solitarii ŽNTS.. Microinjection of L-glutamate into the NTS of anesthetized rats produces hypotension and bradycardia ŽTalman et al., 1980; Gordon and Talman, 1992., a pattern of responses that resembles the cardiovascular responses to baroreflex activation. In spite of substantial evidence in favor of L-glutamate as the putative neurotransmitter of the baroreflex in the NTS, studies by Gordon and Talman
) Corresponding author. Tel.: q55 16 6023015; fax: q55 16 6330017; e-mail: [email protected]
Ž1992. have raised questions about the effective role of L-glutamate in this neurotransmission, because the cardiovascular responses to microinjection of L-glutamate into the NTS were not blocked by microinjection of an excitatory amino acid ŽEAA. receptors antagonist into the NTS, while the bradycardic and hypotensive responses to electrical stimulation of the ADN were abolished. In a previous study ŽMachado and Bonagamba, 1992. we verified that microinjection of L-glutamate into the NTS of unanesthetized rats produces a pressor response that resembles the cardiovascular responses to the chemoreflex activation, while the microinjection of L-glutamate into the NTS of the same animal under chloralose or urethane anethesia produced hypotension and bradycardia. Studies by Colombari et al. Ž1996. have shown that the pressor responses to microinjection of L-glutamate into the commissural NTS of unanesthetized rats were converted to a depressor response when a lesion was made in the midline of the commissural NTS and also that the pressor response to chemoreflex activation was eliminated after the lesion.
0165-1838r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 1 3 7 - 4
44
H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
The pressor response to L-glutamate microinjection into the commissural NTS is sympathetic in origin because treatment of the rats with prazosin, an a1 adrenoceptor antagonist, almost abolished this response ŽColombari et al., 1994.. There is anatomical and functional evidence of a direct projection from the NTS to the RVLM ŽRoss et al., 1985; Urbanski and Sapru, 1988; Aicher et al., 1996.. Therefore, it is possible that microinjection of L-glutamate into the NTS of unanesthetized rats activates the sympatho-excitatory projection to the RVLM, which may be linked to the neural pathways of the chemoreflex. For these reasons, the present study aims to Ža. evaluate if the pressor response to microinjection of L-glutamate into the NTS is or not restricted to the excitation of the ipsilateral RVLM and Žb. verify the role of EAA receptors in the processing of this neurotransmission in the RVLM. To achieve this goal L-glutamate was microinjected into the NTS before and after the microinjection of kynurenic acid, a non-selective EAA receptors antagonist, into the ipsilateral RVLM of conscious freely moving rats. 2. Methods Male Wistar rats weighing 240–260 g were used in the present study. Four days before the experiments, under pentobarbital sodium anesthesia Ž40 mgrkg, i.p., Sigma, USA., the rats were placed in a stereotaxic apparatus ŽDavid Kopf, Tujunga, CA, USA. and two guide cannulas Ž22 gauge, Small Parts, FL, USA. were implanted in the direction of the NTS and RVLM according to the technique of Michelini and Bonagamba Ž1988. and the stereotaxic coordinates of the atlas of Paxinos and Watson Ž1986.. The stereotaxic coordinates for the guide cannulas
were 0.5 mm lateral to the midline, 14 mm caudal to the bregma and 7.9 mm below the skull surface for the NTS and 2.0 mm lateral to the midline, 12.5 mm caudal to the bregma and 6.5 mm below the skull surface for the RVLM. The needles Ž33 gauge, Small Parts. used for microinjection were 1.5 ŽNTS. and 3.5 mm ŽRVLM. longer than the guide cannula and connected by PE-10 tubing to a 1-ml syringe ŽHamilton, Reno, NV.. One day before the experiments, under ether 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 pulsatile arterial pressure ŽPAP., MAP and heart rate ŽHR.. This catheter was tunnelled and exteriorized through the back of the neck to be connected to the recorder system Ža pressure transducer model CDX III, COBE Laboratories, Lakewood, CO, USA. and a Narcotrace 80 Physiological recorder ŽNarco Bio-Systems, Austin, TX, USA.. HR was measured with a Narco-Biotachometer Coupler Žmodel 7302., which is part of the recorder system. The changes in MAP and HR in response to microinjection of L-glutamate into the NTS before and after microinjection of kynurenic acid into the RVLM were considered at the peak of responses. The drugs microinjected into the NTS and RVLM were diluted in saline Ž0.9% NaCl. and all microinjections were performed with a volume of 100 nl. The solutions were freshly dissolved in saline and sodium bicarbonate was added to adjust the pH to the 7.0 to 7.4 range. The needles for microinjection were carefully inserted into the guide cannulas and the injections were manually performed. The experimental protocol consisted of microinjections into the NTS or RVLM at intervals of 15 min. Microinjec-
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 microinjections of saline Ž100 nl. into NTS and RVLM; L-glutamate ŽL-glu. Ž1 nmolr100 nl. into RVLM and NTS; kynurenic acid ŽKYN. Ž2 nmolr100 nl. into the RVLM and L-glu into the NTS 15 min after microinjection of kynurenic acid into the RVLM. The time interval between microinjections into the NTS or RVLM was at least 10 min.
H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
45
tions of saline were first performed into the NTS and RVLM as a vehicle control, followed by microinjection of L-glutamate Ž1 nmolr100 nl. into the RVLM for functional identification of this area. After 15 min, when the MAP and HR were back to baseline levels, L-glutamate Ž1 nmolr100 nl. was microinjected into the NTS before and 15 and 90 min after microinjection of kynurenic acid Ž2 nmolr100 nl. into the ipsilateral RVLM. The last microinjection of L-glutamate into the NTS was performed in order to evaluate the reversibility of the blockade. At the end of the experiments 100 nl of 2% Evan’s blue was microinjected into the NTS and RVLM for identification of the sites of microinjection. The animals were then submitted to intracardiac perfusion with saline followed by 10% buffered formalin under ether anesthesia. The brains were removed and stored in buffered formalin for 2 days, and serial coronal Ž10 mm. sections were cut and stained by the Nissl method. Only the rats in which the sites of microinjection were located into the NTS and RVLM were used for data analysis. Values are reported as means " SEM and statistical analysis was performed using one-way analysis of variance ŽANOVA. for repeated measurements followed by Student’s paired t-test. Differences were considered significant at the level of P - 0.05.
3. Results Fig. 2. Changes in heart rate Ž DHR, upper panel. and mean arterial pressure Ž DMAP, lower panel. in response to microinjections of Lglutamate Ž1 nmolr100 nl. into the lateral commissural NTS before Žcontrol, CON. and 15 and 90 min after microinjection of kynurenic acid ŽKYN. into the RVLM of unanesthetized rats Ž ns8.. Ž). Different compared to the responses to control microinjections Ž P - 0.05..
Fig. 1 shows the tracings of one rat representative of the group Ž n s 8. that received sequential microinjections of saline Žvehicle. into the NTS and RVLM, L-glutamate Ž1 nmolr100 nl. into the RVLM and L-glutamate Ž1 nmolr100 nl. into the commissural NTS before Žcontrol.
Fig. 3. Photomicrograph of coronal sections of the brainstem showing a typical site of microinjection into the lateral commissural NTS Žapproximately 13.8 mm caudal to the bregma.. Arrow show the site of microinjections ŽNissl, 32X..
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H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
Fig. 4. Photomicrograph of coronal section of the brainstem showing a typical site of microinjection into the RVLM Žapproximately 12.8 mm caudal to the bregma.. Arrow show the site of microinjections ŽNissl 32X..
and 15 min after microinjection of kynurenic acid Ž2 nmolr100 nl. into the ipsilateral RVLM. The control microinjections of L-glutamate into the NTS and the RVLM produced a pressor response with bradycardia. The tracings of Fig. 1 show that the major changes produced by microinjection of L-glutamate into the NTS are a rise in pressure and bradycardia, as shown previously by Colombari et al. Ž1994.. The pressor and bradycardic responses to Lglutamate into the NTS, 15 min after kynurenic acid microinjection into the RVLM, were almost abolished. However, 90 min after microinjection of kynurenic acid both pressor and bradycardic responses to L-glutamate microinjected into the NTS were back to the control level, indicating the reversibility of the blockade ŽFig. 2.. Fig. 2 summarizes the data related to the effect of kynurenic acid into the RVLM on the cardiovascular responses produced by microinjection of L-glutamate into the NTS of this group of rats. The control microinjection of L-glutamate into the NTS produced an increase in pressure Žq24 " 3 mm Hg. and bradycardia Žy101" 10 bpm. while the pressor Žq6 " 3 mm Hg. and bradycardic responses Žy3 " 12 bpm. to L-glutamate into the NTS were almost abolished 15 min after kynurenic acid microinjection into the RVLM. The pressor Žq23 " 6 mm Hg. and bradycardic Žy93 " 16 bpm. responses to L-glutamate 90 min later were not statistically different in relation to the control responses. Microinjection of saline Žq1 " 5 mm Hg. or kynurenic acid Žq7 " 6 mm Hg. into the RVLM produced a negligible effect while L-glutamate produced a significant increase in MAP Žq33 " 7 mm Hg.. The changes in HR produced by microinjections of Lglutamate Žy9 " 28 bpm. and kynurenic acid Žy11 " 8 bpm. into the RVLM did not differ significantly from those produced by saline Žy16 " 7 bpm.. Misplaced mi-
croinjections of L-glutamate into areas adjacent to the NTS produced no changes in MAP, while misplaced microinjections of kynurenic acid into areas adjacent to RVLM in rats with effective microinjection of L-glutamate into the ipsilateral NTS produced no blockade of the pressor response. Fig. 3 is a photomicrograph of a coronal section of the brainstem of one rat representative of the group showing the site of microinjection into the commissural NTS at the level of the rostral edge of the area postrema. Fig. 4 is a photomicrograph of a coronal section of the brainstem of the same rat as presented in Fig. 3 showing the microinjection site in the RVLM located approximately 13.8 mm caudal to the bregma and ipsilateral to the microinjection site in the NTS.
4. Discussion The present study shows that the pressor responses produced by unilateral microinjection of L-glutamate into the NTS is dependent on the ipsilateral RVLM and also that the neurotransmission of this projection from the NTS to the RVLM is mediated by EAA receptors. The pressor and bradycardic responses induced by L-glutamate microinjection into the NTS of unanesthetized rats are in accordance with our previous studies ŽMachado and Bonagamba, 1992. showing that microinjection of L-glutamate into the NTS of unanesthetized rats produced opposite responses in arterial pressure in comparison with microinjection of Lglutamate into the NTS of the same rat under chloralose or urethane anesthesia. In another study from our laboratory, we also showed that the pressor response was blocked in a dose-dependent manner by kynurenic acid microinjected
H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
into the NTS, indicating that this sympatho-excitatory response Žpressor response. at the NTS level is mediated by EAA receptors ŽColombari et al., 1994.. An important question related to the pressor response to L-glutamate into the NTS is about the possible involvement of the RVLM in this response. It is well known that neurons in the RVLM are critical in the generation of the sympathetic vasomotor tone ŽDampney et al., 1982; Lipski et al., 1996. but no studies have addressed previously whether or not pressor response to L-glutamate into the NTS was dependent on EAA receptors in the ipsilateral RVLM. The findings of the present study suggest that the excitation of RVLM neurons is due to an activation of direct projections from NTS to RVLM or due to activation of projections from the NTS to other areas of the forebrain Žparabrachial and hypothalamic areas, for example. which presents secondary projections to the RVLM ŽKiely and Gordon, 1994., or both. Our findings are supported by a previous study of Urbanski and Sapru Ž1988. showing that after blockade of the neurons in the caudal ventrolateral medulla ŽCVLM., the microinjections of L-glutamate into the NTS produced a pressor response instead of the control depressor response. In spite of the study by Agarwal and Calaresu Ž1990. suggesting a monosynaptic connection between NTS and RVLM, and the studies by Ross et al. Ž1985. and Aicher et al. Ž1996. reporting the existence of bilateral projections from NTS to the ventrolateral medulla, the functional data of the present study do not allow us to confirm which pathway is involved in this sympatho-excitatory projection. However, the data strongly support the concept that the ipsilateral but not the contralateral RVLM is integral to this sympatho-excitatory pathways. Since in the present study microinjection of L-glutamate into the NTS of unanesthetized rats produced a pressor response similar to that observed with chemoreflex activation ŽHaibara et al., 1995., we may suggest that chemoreflex activation and microinjection of L-glutamate into the NTS activate the same neural pathway from the NTS to the RVLM. Therefore, the findings of the present study have important implications for the understanding of the central neural pathways of the sympatho-excitatory component of the chemoreflex. A study by Koshiya et al. Ž1993. showed that blockade of the RVLM abolished the sympathetic component of the chemoreflex. In addition, Amano et al. Ž1994. showed that microinjection of kynurenic acid into the RVLM ipsilateral to the excited carotid body chemoreceptor abolished the pressor response of the chemoreflex, but produced no effect on the pressor response when the contralateral carotid body chemoreflex was activated. These evidences together indicate that excitatory amino acid receptors in the RVLM are involved in the pressor response of the chemoreflex, and suggest that the activation of the sympatho-excitatory pathways in the NTS by carotid chemoreflex or by L-glutamate microinjection into the NTS involves only neurons of the ipsilateral RVLM.
47
In addition to a pressor response, the microinjection of into the NTS produced a bradycardic response which was also blocked by kynurenic acid microinjected into the RVLM. Several studies indicate that the nucleus ambiguus, located dorsal to RVLM, constitutes the site of origin of the parasympathetic pre-ganglionic neurons to the heart ŽKalia and Sullivan, 1982; Stuesse and Fish, 1984. and seems to be part of the baroreflex pathway ŽLoewy et al., 1990.. The blockade of the bradycardic response to L-glutamate in the present study may be explained by the diffusion of kynurenic acid from the RVLM to the nucleus ambiguus, considering that the microinjection of 100 nl into the RVLM presented a spreading of approximately 500 mm in the antero-posterior coordinates. A similar blockade of the bradycardic response to L-glutamate into the NTS was obtained by Urbanski and Sapru Ž1988. when the CVLM was blocked with kynurenic acid. In addition, studies by Nosaka et al. Ž1979. documented that cardiac preganglionic neurons were also located in the RVLM. Therefore, it is possible that microinjection of kynurenic acid into the RVLM also blocked these cardiac preganglionic neurons. In conclusion, the present study shows that pressor response to microinjection of L-glutamate into the NTS is dependent on the ipsilateral RVLM and also that this response is mediated by EAA receptors. Whether or not this sympatho-excitatory projection from NTS to RVLM activated by microinjection of L-glutamate is part of the neural pathways of the chemoreflex is still a matter for further investigation. L-glutamate
Acknowledgements We thank Leni G.H. Bonagamba for her excellent technical assistance and Rubens F. de 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 Tec´ Ž . nologico CNPQ and Programa Nacional para os Nucleos ´ ´ ŽPRONEX I.. de Excelencia ˆ
References Agarwal, S.K., Calaresu, F.R., 1990. Reciprocal connections between nucleus tractus solitarii and rostral ventrolateral medulla. Brain Res. 523, 305–308. Aicher, S.A., Saravay, R.H., Cravo, S., Jeske, I., Morrison, S.F., Reis, D.J., Milner, T.A., 1996. Monosynaptic projections from the nucleus tractus solitarii to C1 adrenergic neurons in the rostral ventrolateral medulla: comparison with input from the caudal ventrolateral medulla. J. Comp. Neurol. 373, 62–75. Amano, M., Asari, T., Kubo, T., 1994. Excitatory amino acid receptors in the rostral ventrolateral medulla mediate hypertension induced by carotid body chemoreceptor stimulation. Naunyn-Schmiedeberg’s Arch. Pharmacol. 349, 549–554.
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Chianca, D.A. Jr., Machado, B.H., 1996. Microinjection of NMDA antagonist into the NTS of conscious rats blocks the Bezold–Jarisch reflex. Brain Res. 718, 185–188. Colombari, E., Bonagamba, L.G.H., Machado, B.H., 1994. Mechanisms of pressor and bradycardic responses to L-glutamate microinjected into the NTS of conscious rats. Am. J. Physiol. 266, R730–R738, ŽRegulatory Integrative Comp. Physiol. 35.. Colombari, E., Menani, J.V., Talman, W.T., 1996. Commissural NTS contributes to pressor responses to glutamate injected into the medial NTS of awake rats. Am. J. Physiol. 270, R1220–R1225, ŽRegulatory Integrative Comp. Physiol. 39.. Dampney, R.A.L., Goodchil, A.K., Robertson, L.G., Montgomery, W., 1982. Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study. Brain Res. 249, 223–235. Gordon, F.J., Talman, W.T., 1992. Role of excitatory amino acids and their receptors in bulbospinal control of cardiovascular function. In: Kunos, G., Ciriello, J. ŽEds.., Central Neural Mechanisms in Cardiovascular Regulation. Birkhauser, Boston, pp. 209–225. Haibara, A.S., Colombari, E., Chianca, D.A. Jr., Bonagamba, L.G.H., Machado, B.H., 1995. NMDA receptors in NTS are involved in bradycardic but not in pressor response of chemoreflex. Am. J. Physiol 269, H1421–H1427, ŽHeart Circ. Physiol. 38.. Kalia, M., Sullivan, J.M., 1982. Brainstem projections of sensory and motor components of the vagus in the rat. J. Comp. Neurol. 211, 248–264. Kiely, J.M., Gordon, F.J., 1994. Role of rostral ventrolateral medulla in centrally mediated pressor responses. Am. J. Physiol. 267, H1549– H1556, ŽHeart Circ. Physiol., 36.. Koshiya, N., Huangfu, D., Guyenet, P.G., 1993. Ventrolateral medulla and sympathetic chemoreflex in the rat. Brain Res. 609, 174–184. Lipski, J., Kanjhan, R., Kruzewska, B., Rong, W., 1996. Properties of presympathetic neurones in the rostral ventrolateral medulla in the rat: an intracellular study in vivo. J. Physiol. 490, 729–744.
Loewy, A.D., Spyer, K.M., 1990. Vagal preganglionic neurons. In: Loewy, A.D., Spyer, K.M. ŽEds.., Central Regulation of Autonomic Functions. Oxford University Press, pp. 68–87. Machado, B.H., Bonagamba, L.G.H., 1992. Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats. Brain Res. 576, 131–138. Michelini, L.C., Bonagamba, L.G.H., 1988. Baroreceptor reflex modulation by vasopressin microinjected into de nucleus tractus solitarii of conscious rats. Hypertension 11, I75–I79, I. Nosaka, S., Yamamoto, T., Yasunaga, K., 1979. Localization of vagal cardioinhibitory preganglionic neurons within rat brain stem. J. Comp. Neurol. 186, 79–92. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, Sydney. Ross, C.A., Ruggiero, D.A., Reis, D.J., 1985. Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J. Comp. Neurol. 242, 511–534. Stuesse, S.L., Fish, S.E., 1984. Projections to the cardioinhibitory region of nucleus ambiguus of rat. J. Comp. Neurol. 229, 271–278. Talman, W.T., Perrone, M.H., Reis, D.J., 1980. Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers. Science 209, 813–814. Urbanski, R.W., Sapru, H.N., 1988. Evidence for a sympathoexcitatory pathway from the nucleus tractus solitarii to the ventrolateral medullary pressor area. J. Auton. Nerv. Sist. 23, 161–174. Vardhan, A., Kachroo, A., Sapru, H.N., 1993. Excitatory amino acid receptors in commissural nucleus of the NTS mediate carotid chemoreceptor responses. Am. J. Physiol. 264, R41–R50, ŽRegulatory Integrative Comp. Physiol., 33.. Zhang, W., Mifflin, S., 1993. Excitatory amino acid receptors within NTS mediate arterial chemoreceptor reflexes in rats. Am. J. Physiol. 265, H770–H773, ŽHeart Circ. Physiol., 34..
Involvement of the ipsilateral rostral ventrolateral medulla in the pressor response to L-glutamate microinjection into the nucleus tractus solitarii of awake rats Helder Mauad b, Benedito H. Machado ´ a
a,)
Department of Physiology, School of Medicine of Ribeirao ˜ Preto, UniÕersity of Sao ˜ Paulo, 14049-900, Ribeirao ˜ Preto, SP, Brazil Department of Physiological Sciences, Biomedical Center, Federal UniÕersity of Espirito Santo, 29040-090, Vitoria, ´ ES, Brazil
b
Received 15 April 1998; accepted 27 July 1998
Abstract Microinjection of L-glutamate into the lateral commissural nucleus tractus solitarii ŽNTS. of unanesthetized rats evokes increases in mean arterial pressure ŽMAP. and a bradycardia. In a previous study we verified that this increase in MAP is mediated sympathetically because prazosin Živ. blocks this response. The aim of the present study was to evaluate the role of the rostral ventrolateral medulla ŽRVLM. in the pressor response produced by L-glutamate microinjected into the NTS of unanesthetized rats. L-Glutamate was microinjected into the NTS before and 15 and 90 min after microinjection of kynurenic acid into the ipsilateral RVLM. Pressor Žq24 " 3 vs. q6 " 3 mm Hg. and bradycardic Žy101" 10 vs. y3 " 12 bpm. responses to L-glutamate microinjected into the NTS Ž n s 8. were almost abolished 15 min after microinjection of kynurenic acid into the RVLM when compared with control responses. Both pressor Žq23 " 6 mm Hg. and bradycardic Žy93 " 16 bpm. responses to L-glutamate into the NTS returned to control values 90 min after microinjection of kynurenic acid into the RVLM. These data indicate that the pressor response to L-glutamate into the NTS is essentially dependent on the ipsilateral RVLM and also that this sympatho-excitatory response is mediated by excitatory amino acid receptors in RVLM neurons. q 1998 Elsevier Science B.V. All rights reserved. Keywords: EAA receptors; Chemoreflex; Cardiovascular regulation; Ionotropic receptors; Sympatho-excitation; Neurotransmitter
1. Introduction Several studies have indicated that the excitatory amino acid L-glutamate seems to play a key role in the neurotransmission of the baro- ŽTalman et al., 1980; Gordon and Talman, 1992; Machado and Bonagamba, 1992; Colombari et al., 1994., chemo- ŽVardhan et al., 1993; Zhang and Mifflin, 1993; Haibara et al., 1995. and Bezold–Jarisch reflex ŽChianca and Machado, 1996. in the nucleus tractus solitarii ŽNTS.. Microinjection of L-glutamate into the NTS of anesthetized rats produces hypotension and bradycardia ŽTalman et al., 1980; Gordon and Talman, 1992., a pattern of responses that resembles the cardiovascular responses to baroreflex activation. In spite of substantial evidence in favor of L-glutamate as the putative neurotransmitter of the baroreflex in the NTS, studies by Gordon and Talman
) Corresponding author. Tel.: q55 16 6023015; fax: q55 16 6330017; e-mail: [email protected]
Ž1992. have raised questions about the effective role of L-glutamate in this neurotransmission, because the cardiovascular responses to microinjection of L-glutamate into the NTS were not blocked by microinjection of an excitatory amino acid ŽEAA. receptors antagonist into the NTS, while the bradycardic and hypotensive responses to electrical stimulation of the ADN were abolished. In a previous study ŽMachado and Bonagamba, 1992. we verified that microinjection of L-glutamate into the NTS of unanesthetized rats produces a pressor response that resembles the cardiovascular responses to the chemoreflex activation, while the microinjection of L-glutamate into the NTS of the same animal under chloralose or urethane anethesia produced hypotension and bradycardia. Studies by Colombari et al. Ž1996. have shown that the pressor responses to microinjection of L-glutamate into the commissural NTS of unanesthetized rats were converted to a depressor response when a lesion was made in the midline of the commissural NTS and also that the pressor response to chemoreflex activation was eliminated after the lesion.
0165-1838r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 1 3 7 - 4
44
H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
The pressor response to L-glutamate microinjection into the commissural NTS is sympathetic in origin because treatment of the rats with prazosin, an a1 adrenoceptor antagonist, almost abolished this response ŽColombari et al., 1994.. There is anatomical and functional evidence of a direct projection from the NTS to the RVLM ŽRoss et al., 1985; Urbanski and Sapru, 1988; Aicher et al., 1996.. Therefore, it is possible that microinjection of L-glutamate into the NTS of unanesthetized rats activates the sympatho-excitatory projection to the RVLM, which may be linked to the neural pathways of the chemoreflex. For these reasons, the present study aims to Ža. evaluate if the pressor response to microinjection of L-glutamate into the NTS is or not restricted to the excitation of the ipsilateral RVLM and Žb. verify the role of EAA receptors in the processing of this neurotransmission in the RVLM. To achieve this goal L-glutamate was microinjected into the NTS before and after the microinjection of kynurenic acid, a non-selective EAA receptors antagonist, into the ipsilateral RVLM of conscious freely moving rats. 2. Methods Male Wistar rats weighing 240–260 g were used in the present study. Four days before the experiments, under pentobarbital sodium anesthesia Ž40 mgrkg, i.p., Sigma, USA., the rats were placed in a stereotaxic apparatus ŽDavid Kopf, Tujunga, CA, USA. and two guide cannulas Ž22 gauge, Small Parts, FL, USA. were implanted in the direction of the NTS and RVLM according to the technique of Michelini and Bonagamba Ž1988. and the stereotaxic coordinates of the atlas of Paxinos and Watson Ž1986.. The stereotaxic coordinates for the guide cannulas
were 0.5 mm lateral to the midline, 14 mm caudal to the bregma and 7.9 mm below the skull surface for the NTS and 2.0 mm lateral to the midline, 12.5 mm caudal to the bregma and 6.5 mm below the skull surface for the RVLM. The needles Ž33 gauge, Small Parts. used for microinjection were 1.5 ŽNTS. and 3.5 mm ŽRVLM. longer than the guide cannula and connected by PE-10 tubing to a 1-ml syringe ŽHamilton, Reno, NV.. One day before the experiments, under ether 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 pulsatile arterial pressure ŽPAP., MAP and heart rate ŽHR.. This catheter was tunnelled and exteriorized through the back of the neck to be connected to the recorder system Ža pressure transducer model CDX III, COBE Laboratories, Lakewood, CO, USA. and a Narcotrace 80 Physiological recorder ŽNarco Bio-Systems, Austin, TX, USA.. HR was measured with a Narco-Biotachometer Coupler Žmodel 7302., which is part of the recorder system. The changes in MAP and HR in response to microinjection of L-glutamate into the NTS before and after microinjection of kynurenic acid into the RVLM were considered at the peak of responses. The drugs microinjected into the NTS and RVLM were diluted in saline Ž0.9% NaCl. and all microinjections were performed with a volume of 100 nl. The solutions were freshly dissolved in saline and sodium bicarbonate was added to adjust the pH to the 7.0 to 7.4 range. The needles for microinjection were carefully inserted into the guide cannulas and the injections were manually performed. The experimental protocol consisted of microinjections into the NTS or RVLM at intervals of 15 min. Microinjec-
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 microinjections of saline Ž100 nl. into NTS and RVLM; L-glutamate ŽL-glu. Ž1 nmolr100 nl. into RVLM and NTS; kynurenic acid ŽKYN. Ž2 nmolr100 nl. into the RVLM and L-glu into the NTS 15 min after microinjection of kynurenic acid into the RVLM. The time interval between microinjections into the NTS or RVLM was at least 10 min.
H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
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tions of saline were first performed into the NTS and RVLM as a vehicle control, followed by microinjection of L-glutamate Ž1 nmolr100 nl. into the RVLM for functional identification of this area. After 15 min, when the MAP and HR were back to baseline levels, L-glutamate Ž1 nmolr100 nl. was microinjected into the NTS before and 15 and 90 min after microinjection of kynurenic acid Ž2 nmolr100 nl. into the ipsilateral RVLM. The last microinjection of L-glutamate into the NTS was performed in order to evaluate the reversibility of the blockade. At the end of the experiments 100 nl of 2% Evan’s blue was microinjected into the NTS and RVLM for identification of the sites of microinjection. The animals were then submitted to intracardiac perfusion with saline followed by 10% buffered formalin under ether anesthesia. The brains were removed and stored in buffered formalin for 2 days, and serial coronal Ž10 mm. sections were cut and stained by the Nissl method. Only the rats in which the sites of microinjection were located into the NTS and RVLM were used for data analysis. Values are reported as means " SEM and statistical analysis was performed using one-way analysis of variance ŽANOVA. for repeated measurements followed by Student’s paired t-test. Differences were considered significant at the level of P - 0.05.
3. Results Fig. 2. Changes in heart rate Ž DHR, upper panel. and mean arterial pressure Ž DMAP, lower panel. in response to microinjections of Lglutamate Ž1 nmolr100 nl. into the lateral commissural NTS before Žcontrol, CON. and 15 and 90 min after microinjection of kynurenic acid ŽKYN. into the RVLM of unanesthetized rats Ž ns8.. Ž). Different compared to the responses to control microinjections Ž P - 0.05..
Fig. 1 shows the tracings of one rat representative of the group Ž n s 8. that received sequential microinjections of saline Žvehicle. into the NTS and RVLM, L-glutamate Ž1 nmolr100 nl. into the RVLM and L-glutamate Ž1 nmolr100 nl. into the commissural NTS before Žcontrol.
Fig. 3. Photomicrograph of coronal sections of the brainstem showing a typical site of microinjection into the lateral commissural NTS Žapproximately 13.8 mm caudal to the bregma.. Arrow show the site of microinjections ŽNissl, 32X..
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H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
Fig. 4. Photomicrograph of coronal section of the brainstem showing a typical site of microinjection into the RVLM Žapproximately 12.8 mm caudal to the bregma.. Arrow show the site of microinjections ŽNissl 32X..
and 15 min after microinjection of kynurenic acid Ž2 nmolr100 nl. into the ipsilateral RVLM. The control microinjections of L-glutamate into the NTS and the RVLM produced a pressor response with bradycardia. The tracings of Fig. 1 show that the major changes produced by microinjection of L-glutamate into the NTS are a rise in pressure and bradycardia, as shown previously by Colombari et al. Ž1994.. The pressor and bradycardic responses to Lglutamate into the NTS, 15 min after kynurenic acid microinjection into the RVLM, were almost abolished. However, 90 min after microinjection of kynurenic acid both pressor and bradycardic responses to L-glutamate microinjected into the NTS were back to the control level, indicating the reversibility of the blockade ŽFig. 2.. Fig. 2 summarizes the data related to the effect of kynurenic acid into the RVLM on the cardiovascular responses produced by microinjection of L-glutamate into the NTS of this group of rats. The control microinjection of L-glutamate into the NTS produced an increase in pressure Žq24 " 3 mm Hg. and bradycardia Žy101" 10 bpm. while the pressor Žq6 " 3 mm Hg. and bradycardic responses Žy3 " 12 bpm. to L-glutamate into the NTS were almost abolished 15 min after kynurenic acid microinjection into the RVLM. The pressor Žq23 " 6 mm Hg. and bradycardic Žy93 " 16 bpm. responses to L-glutamate 90 min later were not statistically different in relation to the control responses. Microinjection of saline Žq1 " 5 mm Hg. or kynurenic acid Žq7 " 6 mm Hg. into the RVLM produced a negligible effect while L-glutamate produced a significant increase in MAP Žq33 " 7 mm Hg.. The changes in HR produced by microinjections of Lglutamate Žy9 " 28 bpm. and kynurenic acid Žy11 " 8 bpm. into the RVLM did not differ significantly from those produced by saline Žy16 " 7 bpm.. Misplaced mi-
croinjections of L-glutamate into areas adjacent to the NTS produced no changes in MAP, while misplaced microinjections of kynurenic acid into areas adjacent to RVLM in rats with effective microinjection of L-glutamate into the ipsilateral NTS produced no blockade of the pressor response. Fig. 3 is a photomicrograph of a coronal section of the brainstem of one rat representative of the group showing the site of microinjection into the commissural NTS at the level of the rostral edge of the area postrema. Fig. 4 is a photomicrograph of a coronal section of the brainstem of the same rat as presented in Fig. 3 showing the microinjection site in the RVLM located approximately 13.8 mm caudal to the bregma and ipsilateral to the microinjection site in the NTS.
4. Discussion The present study shows that the pressor responses produced by unilateral microinjection of L-glutamate into the NTS is dependent on the ipsilateral RVLM and also that the neurotransmission of this projection from the NTS to the RVLM is mediated by EAA receptors. The pressor and bradycardic responses induced by L-glutamate microinjection into the NTS of unanesthetized rats are in accordance with our previous studies ŽMachado and Bonagamba, 1992. showing that microinjection of L-glutamate into the NTS of unanesthetized rats produced opposite responses in arterial pressure in comparison with microinjection of Lglutamate into the NTS of the same rat under chloralose or urethane anesthesia. In another study from our laboratory, we also showed that the pressor response was blocked in a dose-dependent manner by kynurenic acid microinjected
H. Mauad, B.H. Machador Journal of the Autonomic NerÕous System 74 (1998) 43–48
into the NTS, indicating that this sympatho-excitatory response Žpressor response. at the NTS level is mediated by EAA receptors ŽColombari et al., 1994.. An important question related to the pressor response to L-glutamate into the NTS is about the possible involvement of the RVLM in this response. It is well known that neurons in the RVLM are critical in the generation of the sympathetic vasomotor tone ŽDampney et al., 1982; Lipski et al., 1996. but no studies have addressed previously whether or not pressor response to L-glutamate into the NTS was dependent on EAA receptors in the ipsilateral RVLM. The findings of the present study suggest that the excitation of RVLM neurons is due to an activation of direct projections from NTS to RVLM or due to activation of projections from the NTS to other areas of the forebrain Žparabrachial and hypothalamic areas, for example. which presents secondary projections to the RVLM ŽKiely and Gordon, 1994., or both. Our findings are supported by a previous study of Urbanski and Sapru Ž1988. showing that after blockade of the neurons in the caudal ventrolateral medulla ŽCVLM., the microinjections of L-glutamate into the NTS produced a pressor response instead of the control depressor response. In spite of the study by Agarwal and Calaresu Ž1990. suggesting a monosynaptic connection between NTS and RVLM, and the studies by Ross et al. Ž1985. and Aicher et al. Ž1996. reporting the existence of bilateral projections from NTS to the ventrolateral medulla, the functional data of the present study do not allow us to confirm which pathway is involved in this sympatho-excitatory projection. However, the data strongly support the concept that the ipsilateral but not the contralateral RVLM is integral to this sympatho-excitatory pathways. Since in the present study microinjection of L-glutamate into the NTS of unanesthetized rats produced a pressor response similar to that observed with chemoreflex activation ŽHaibara et al., 1995., we may suggest that chemoreflex activation and microinjection of L-glutamate into the NTS activate the same neural pathway from the NTS to the RVLM. Therefore, the findings of the present study have important implications for the understanding of the central neural pathways of the sympatho-excitatory component of the chemoreflex. A study by Koshiya et al. Ž1993. showed that blockade of the RVLM abolished the sympathetic component of the chemoreflex. In addition, Amano et al. Ž1994. showed that microinjection of kynurenic acid into the RVLM ipsilateral to the excited carotid body chemoreceptor abolished the pressor response of the chemoreflex, but produced no effect on the pressor response when the contralateral carotid body chemoreflex was activated. These evidences together indicate that excitatory amino acid receptors in the RVLM are involved in the pressor response of the chemoreflex, and suggest that the activation of the sympatho-excitatory pathways in the NTS by carotid chemoreflex or by L-glutamate microinjection into the NTS involves only neurons of the ipsilateral RVLM.
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In addition to a pressor response, the microinjection of into the NTS produced a bradycardic response which was also blocked by kynurenic acid microinjected into the RVLM. Several studies indicate that the nucleus ambiguus, located dorsal to RVLM, constitutes the site of origin of the parasympathetic pre-ganglionic neurons to the heart ŽKalia and Sullivan, 1982; Stuesse and Fish, 1984. and seems to be part of the baroreflex pathway ŽLoewy et al., 1990.. The blockade of the bradycardic response to L-glutamate in the present study may be explained by the diffusion of kynurenic acid from the RVLM to the nucleus ambiguus, considering that the microinjection of 100 nl into the RVLM presented a spreading of approximately 500 mm in the antero-posterior coordinates. A similar blockade of the bradycardic response to L-glutamate into the NTS was obtained by Urbanski and Sapru Ž1988. when the CVLM was blocked with kynurenic acid. In addition, studies by Nosaka et al. Ž1979. documented that cardiac preganglionic neurons were also located in the RVLM. Therefore, it is possible that microinjection of kynurenic acid into the RVLM also blocked these cardiac preganglionic neurons. In conclusion, the present study shows that pressor response to microinjection of L-glutamate into the NTS is dependent on the ipsilateral RVLM and also that this response is mediated by EAA receptors. Whether or not this sympatho-excitatory projection from NTS to RVLM activated by microinjection of L-glutamate is part of the neural pathways of the chemoreflex is still a matter for further investigation. L-glutamate
Acknowledgements We thank Leni G.H. Bonagamba for her excellent technical assistance and Rubens F. de 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 Tec´ Ž . nologico CNPQ and Programa Nacional para os Nucleos ´ ´ ŽPRONEX I.. de Excelencia ˆ
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