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Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, São Paulo, Brazil
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Abstract |
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-methyl-4-carboxyphenylglycine (MCPG; 5 nmol/50 nL), a non-selective antagonist of metabotropic receptors. The results showing that the cardiovascular responses to microinjection of trans-ACPD into the NTS were blocked by AP-5 indicate that the responses to metabotropic agonists in the NTS involves NMDA receptors.
(Received 6 October 2003;
accepted after revision 9 February 2004; first published online 17 February 2004)
Corresponding author B. H. Machado: Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, São Paulo, Brazil. Email: bhmachad{at}fmrp.usp.br
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Methods |
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Male Wistar rats weighing 290–310 g were obtained from the Animal Care Center of the University of São Paulo, campus of Ribeirão Preto, and kept on a 12 h light–dark circadian cycle, with food and water ad libitum. All the experimental procedures described in this study followed the ethical guidelines principles of the School of Medicine of Ribeirão Preto Animal Care and Ethics Committee.
Surgical procedures
Rats were initially anaesthetized with an intraperitoneal injection of tribromoethanol (250 mg kg–1; Aldrich, Milwaukee, WI, USA) and placed in a stereotaxic apparatus (David Kopf, Tujunga, CA, USA) and bilateral guide cannulas (0.5 mm lateral to midline) were implanted in the direction of the lateral aspect of the commissural NTS according to the coordinates of Paxinos & Watson (1986) and the technique described by Michelini & Bonagamba (1988). During the surgery for guide cannulas implantation, the local anaesthetic lidocaine with vasoconstrictor was injected subcutaneously in the dorsal aspect of the head to reduce the bleeding. Additional tribromoethanol was injected when the rats reacted to frequent toe pinching during the stereotaxic surgery. After the stereotaxic surgery, rats received 0.2 ml (1.2 million U) of veterinary pentabiotic (Fontoura-Wyeth, São Paulo, Brazil) administered subcutaneously.
Although microinjections into the NTS were performed unilaterally, the guide cannulas were implanted in both sides. With this approach the index of positive histology was higher than just unilateral implantation because before the experimental protocol we microinjected L-glutamate into the NTS for a functional identification of the site of microinjection in accordance with our previous studies (Machado & Bonagamba, 1992; Colombari et al. 1994). In case that one side was not responsive to microinjection of L-glutamate we also tried microinjection into the NTS via the second guide cannula.
Three days after the surgery for guide cannulae implantation and one day before the experiments the rats were anaesthetized with tribromoethanol anaesthesia (250 mg kg–1, I.P.), and a catheter (PE-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 (PAP), mean arterial pressure (MAP) and heart rate (HR). A second catheter was inserted into the femoral vein for drug administration. The catheters were tunneled and exteriorized in the back of the neck to be connected to a pressure transducer under conscious freely moving conditions. PAP and MAP were measured with a transducer (Model CDX III, Cobe Laboratories, Lakewood, CO, USA) connected to a physiological recorder (Narcotrace 80; Narco Bio-Systems, Austin, TX, USA). HR was derived from the pulsatile arterial pressure with a Narco biotachometer coupler (Narco Bio-Systems, Model 7302).
Microinjections into the NTS
The needle (33 gauge, Small Parts, Miami Lakes, FL, USA) used for microinjection into the NTS was 1.5 mm longer than the guide cannula and was connected by PE-10 tubing to a 1 µl syringe (Hamilton, Reno, NV, USA). The needle was carefully inserted into the guide cannula, manual injections were performed 30 s later and the volume injected was always 50 nL. In accordance with previous experiments from our laboratory, all rats in the different experimental protocols initially received a unilateral microinjection of L-glutamate (1 nmol/50 nL) for functional identification of the NTS (Machado & Bonagamba, 1992; Colombari et al. 1997) and the experiments involving metabotropic and ionotropic receptor antagonists were initiated at least 15 min after microinjection of L-glutamate into the NTS, when both MAP and HR were back to control levels. The time interval between microinjections into the NTS varied in accordance with the experimental protocol; trans-ACPD was microinjected 10 min after AP-5 while NMDA was microinjected 5 min after -methyl-4-carboxyphenlyglycine (MCPG). These time intervals were determined in accordance with previous studies from our laboratory showing the efficacy of these antagonists (Frigero et al. 2000; Antunes & Machado, 2003). The doses of L-glutamate, NMDA and trans-ACPD used in these experiments correspond to the EC50 determined in previous studies from our laboratory (Colombari et al. 1994; Machado & Bonagamba, 1998; Frigero et al. 2000). At the end of each experiment, 50 nL of Evans' blue (2%) were microinjected into the same sites for histological analysis. The animals were killed with an overdose of thiopental sodium (I.V.) and submitted to intracardiac perfusion with saline followed by 10% buffered formalin. The brains were removed and stored in buffered formalin for 2 days, and serial coronal sections (15 µm thickness) were cut and stained by the Nissl method. Only the rats in which the site of microinjection was located in the lateral aspect of the commissural NTS were used for data analysis. In this case approximately 40% of the implanted rats presented positive histology. We verified in a group of the rats with misplaced microinjection in areas adjacent to the NTS (n= 6) that trans-ACPD produced neglegible effects on the MAP (2 ± 6 mmHg) and HR (–8 ± 9 bpm).
Data analysis
The data obtained in each experimental protocol were analysed statistically by one-way analysis of variance (ANOVA) followed by the Tukey post hoc test. Differences were considered to be significant at P < 0.05. All values are reported as means ±S.E.M.
Drug solutions
The following drugs were used in the experiments: L-glutamate, trans-1-amino-1,3-cyclopentanediocarboxylic acid (trans-ACPD, a glutamatergic metabotropic receptor agonist, RBI, Natick, MA, USA), -methyl-4-carboxyphenylglycine (MCPG, a non-selective glutamatergic metabotropic receptor antagonist, RBI, Natick, MA, USA), N-methyl-D-aspartic acid (a NMDA receptor agonist, RBI, Natick, MA, USA), 2-amino-5-phosphonovaleric acid (AP-5, a selective NMDA receptors antagonist, RBI, Natick, MA, USA) and methyl-atropine (a muscarinic receptor antagonist, Sigma, Saint Louis, MO, USA). The drug solutions were freshly dissolved in 0.9% saline solution and sodium bicarbonate was added to adjust the pH to 7.4.
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Results |
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Figure 1 presents a typical tracing of one rat representative of the group showing the hypotensive and bradycardic response to microinjection of trans-ACPD (250 pmol/50 nL) into the NTS before and 10 and 60 min after microinjection of AP-5 (10 nmol/50 nL) into the same site in the NTS. In addition, the tracings in the figure show that after the hypotensive and bradycardic responses to trans-ACPD were back to control levels, the injection of methyl-atropine (I.V.) blocked both the bradycardic and hypotensive responses to a subsequent microinjection of trans-ACPD into the NTS. Figure 2 summarizes the data and shows that the hypotensive (–64 ± 4 versus–6 ± 4 mmHg) and bradycardic responses (–206 ± 11 versus–21 ± 12 bpm) to microinjection of trans-ACPD into the NTS (n= 8) 10 min after microinjection of AP-5 into the same site were significantly reduced in comparison to the control responses. The antagonism produced by the microinjection of AP-5 was reversible because the cardiovascular responses to microinjection of trans-ACPD were back to control levels 60 min later. Figure 2 also shows that the subsequent injection of methyl-atropine (2 mg kg–1, I.V.) blocked the bradycardic and hypotensive responses to microinjection of trans-ACPD, suggesting that the hypotension was determined mainly by the intense bradycardic response. The blockade of the bradycardic and hypotensive responses to trans-ACPD by methyl-atropine was similar to the findings of a previous study from our laboratory using a different experimental protocol (Antunes & Machado, 2003).
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Antagonism of metabotropic receptors and the cardiovascular responses to microinjection of NMDA into the NTS
Figure 3 presents a typical tracing of one rat representative of the group showing the hypotensive and bradycardic response to microinjection of NMDA (10 pmol/50 nL) into the NTS before and 5 and 30 min after microinjection of MCPG (5 nmol/50 nL) into the same site in the NTS. The data summarized in Fig. 4 show that the hypotensive response to microinjection of NMDA (10 pmol/50 nL) into the NTS at 5 min (–40 ± 5 mmHg) and 30 min (–43 ± 8 mmHg) after microinjection of MCPG (5 nmol/50 nL, n= 8) into the same site did not differ from the control response (–46 ± 6 mmHg). Moreover, the bradycardic response to microinjection of NMDA into the NTS was also not affected by previous microinjection of the MCPG at 5 min (–196 ± 31 bpm) and 30 min (–196 ± 34 bpm) when compared with the control response (–238 ± 20 bpm). Microinjection of NMDA was performed 5 min after MCPG because previous studies from our laboratory (Antunes & Machado, 2003) showed that this dose of MCPG was effective in blocking the response to trans-ACPD even 10 min later.
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Discussion |
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The nucleus of the solitary tract (NTS) plays an important role in the central regulation of cardiovascular function (Gordon & Talman, 1992; Sved & Gordon, 1994). Several studies have suggested that L-glutamate is the neurotransmitter released by the baroreceptor afferents in the NTS (Talman et al. 1980; Ciriello & Calaresu, 1981; Spyer, 1981; Gordon & Talman, 1992). Other studies have shown that ionotropic glutamatergic receptors in the NTS play an important role in neurotransmission and/or neuromodulation of baroreflex at the NTS level (Talman, 1989; Kubo & Kihara, 1988). Leone & Gordon (1989) have shown that pharmacological blockade of an ionotropic excitatory amino acid in the NTS with kynurenate abolishes baroreceptor reflexes but fails to affect the cardiovascular responses evoked by microinjection of L-glutamate into the same site, suggesting the involvement of another class of glutamatergic receptors in the neurotransmission of the baroreflex in NTS neurones (Leone & Gordon, 1989) which were later characterized as the metabotropic receptors.
Pharmacological studies (Pawloski-Dahm & Gordon, 1992) have indicated that microinjection of a metabotropic agonist (trans-ACPD) into the NTS of anaesthetized rats produced cardiovascular changes similar to those produced by L-glutamate (hypotension and bradycardia) and these responses were not affected by previous microinjection of kynurenic acid into the same site. Studies from our laboratory (Machado & Bonagamba, 1998) have shown that unilateral microinjection of trans-ACPD into the NTS of awake rats or in the same animals under anaesthesia produced hypotension and bradycardia. Recently we verified that the gain of the baroreflex was not affected by the blockade of metabotropic receptors in the NTS, suggesting that this class of receptors is not integral to the processing of the parasympathetic component of the baroreflex at the NTS level. Nonetheless, the role of metabotropic receptors in the modulation of the sympathoinhibitory component of the baroreflex in awake rats rats remain to be evaluated.
Considering that (a) microinjection of trans-ACPD into the NTS produced hypotension and bradycardia (Machado & Bonagamba, 1998), and (b) the gain of the baroreflex was significantly reduced by previous microinjection of an NMDA receptor antagonist (AP-5) into the NTS (Frigero et al. 2000) and the bradycardic responses to chemoreflex or Bezold–Jarish reflex activation or to microinjection of L-glutamate into the NTS (Chianca & Machado, 1996; Haibara et al. 1995; Colombari et al. 1997), in the present study we evaluated the possible interaction between a metabotropic receptor agonist (trans-ACPD) and NMDA ionotropic receptors. The data indicate that previous microinjection of AP-5 into the NTS blocked the cardiovascular responses to microinjection of trans-ACPD at the same site. It is possible that trans-ACPD acting on the metabotropic receptors located on the NTS neurones projecting to nucleus ambiguus/dorsal motor nucleus of the vagus may facilitate the activation of NMDA receptors, which generate the parasympathetic response. This suggestion is supported by in vitro studies showing that activation of metabotropic receptor potentiates inward currents mediated by NMDA receptors in hippocampal neurones (Aniksztejn et al. 1991) as well as in the rat dorsal horn (Bleakman et al. 1992).
The data from a specific experimental protocol of the present study, as well as from a different experimental protocol in a recent study from our laboratory (Antunes & Machado, 2003), show that the hypotension produced by microinjection of trans-ACPD into the NTS was secondary to the intense bradycardic response because administration of methyl-atropine blocked the bradycardia and the hypotensive response. These data obtained in awake rats suggest that trans-ACPD activates mainly neurones involved in the parasympathetic component at the NTS level, probably by indirect excitation of NMDA receptors, considering that various studies from our laboratory have shown that the parasympathetic component of the cardiovascular reflexes at the NTS level is mediated mainly by NMDA receptors (Haibara et al. 1995; Chianca & Machado, 1996; Colombari et al. 1997; Frigero et al. 2000; Machado, 2001). Considering the studies by Foley et al. (1999) showing that activation of metabotropic receptors in the NTS of anaesthetized rats elicited bradycardia and sympathoinhibition, we cannot rule out the possibility that in awake rats the sympathoinhibition may also occur. However, in awake rats it is possible that the cardiovascular reflexes compensate the expected fall in arterial pressure due to sympathoinhibition, which may not occur in rats under anaesthesia.
With respect to the interaction of metabotropic and ionotropic receptors at the NTS level there is some evidence supporting this possibility. It is possible that the cardiovascular responses to microinjection of trans-ACPD into the NTS are produced by activation of presynaptic metabotropic receptors, which may facilitate the release of L-glutamate (Glaum & Miller, 1993). Studies by Hay & Hasser (1998), measuring the synaptic terminal exocytosis during stimulation of nodose ganglion neurones in vitro, showed that depolarization-induced exocytosis in baroreceptors terminals was inhibited by L-2-amino-4-phosphonobutyric acid (L-AP4), a type III metabotropic glutamate receptor agonist, suggesting that presynaptic metabotropic receptor activation, in this case, inhibits the vesicle release. Specifically in relation to a possible interaction of trans-ACPD with NMDA receptors, studies by Aniksztejn et al. (1991) showed that the activation of metabotropic receptors increase the responses mediated by NMDA receptors but not the responses mediated by non-NMDA receptors.
Studies by Leone & Gordon (1989) and Pawloski-Dahm & Gordon (1992) showed that the cardiovascular response to microinjection of L-glutamate into the NTS of anaesthetized rats still produced bradycardia and hypotension after the selective blockade of ionotropic glutamate receptors, and the authors suggested that the remaining effect was due to the activation of metabotropic receptors. In accordance with these previous findings, the blockade of NMDA receptors in the present study should not affect the response to metabotropic activation by microinjection of trans-ACPD. However, the data of the present study clearly demonstrate that the cardiovascular responses to microinjection of trans-ACPD into the NTS of awake rats were almost blocked by AP-5. Recent studies by Foley et al. (1999) and Viard & Sapru (2002), also performed in anaesthetized rats, showed that the antagonism of ionotropic glutamate receptors in the NTS had no effect on the subsequent activation of metabotropic receptors, different from the findings of the present study. In all these cases the opposite findings may be explained by the important differences from the experimental approaches used by these different laboratories, which include the use of anaesthetized or unanaesthetized rats, the anaesthetic, the level of anaesthesia, the baseline level of heart rate and arterial pressure, the different doses of agonists and antagonists, the different volume microinjected into the NTS and mainly with respect to the integrity of the cardiovascular reflexes which are not completely preserved in anaesthetized animals.
Previous studies from our laboratory (Haibara et al. 1999; Antunes & Machado, 2003) documented that the dose of MCPG used in the present study was effective in producing a significant reduction in the cardiovascular response to microinjection of trans-ACPD into the NTS. With respect to the effect of metabotropic receptor antagonism (MCPG) on the activation of NMDA receptors, the data show that the cardiovascular responses (hypotension and bradycardia) to microinjection of NMDA into the NTS were not affected by local microinjection of MCPG. This finding indicates that the effect of MCPG on the cardiovascular responses to trans-ACPD is not related to the blockade of NMDA receptors and suggests that the interaction of the metabotropic and NMDA receptors may occur at pre or postsynaptic levels or at the level of interneurones but not directly on NMDA receptors.
We conclude that cardiovascular responses to microinjection of trans-ACPD into the NTS may involve an interaction with glutamatergic ionotropic NMDA receptors. However, the specific mechanism of which remains as an important question that requires further investigation.
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References |
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MAP, top panel) and heart rate (
