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Autonomic and respiratory responses to microinjection of l -glutamate into the commissural subnucleus of the NTS in the working heart–brainstem preparation of the rat
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Research Report
Autonomic and respiratory responses to microinjection of L-glutamate into the commissural subnucleus of the NTS in the working heart–brainstem preparation of the rat Valdir A. Braga, Vagner R. Antunes, Benedito H. Machado⁎ Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Changes in heart rate (HR), thoracic sympathetic nerve activity (tSNA) and frequency of
Accepted 20 March 2006
phrenic nerve discharge (PND) in response to microinjection of L-glutamate before and
Available online 16 May 2006
after local microinjection of ionotropic or metabotropic glutamate receptors antagonists into the commissural subnucleus of the NTS (comNTS) were investigated. The
Keywords:
experiments were performed in an in situ unanesthetized decerebrated working
L-glutamate
heart–brainstem preparation (WHBP), and the main findings were as follows: (a)
Ionotropic receptor
microinjection of increasing concentrations of L-glutamate (5, 25, 50, 250 and 500 mM)
Metabotropic receptor
into the comNTS produced bradycardia, increase in tSNA and reduction in the
Phrenic nerve discharge
frequency of the PND in a concentration-dependent manner; (b) both bradycardia and
Sympathetic nerve activity
increase in tSNA were almost abolished by kynurenic acid (KYN, 250 mM, a nonselective
Heart rate
ionotropic glutamate receptor antagonist); (c) the reduction in the frequency of the PND was reversed to an increase in the frequency of the PND after KYN and this increase was blocked by the sequential microinjection of MCPG (100 mM, a nonselective metabotropic glutamate receptor antagonist); and (d) microinjection of increasing concentrations of trans-ACPD (0.5, 1.0, 2.5, 5.0 and 10 mM, a metabotropic glutamate receptor agonist), elicited bradycardia and increase in the frequency of the PND in a concentration-dependent manner, which were blocked by MCPG. Taken together, these data indicate that
L-glutamate
and its ionotropic receptors are involved in the
sympathoexcitatory, bradycardic and reduction in the frequency of the PND responses whereas/although its metabotropic receptors are involved in the bradycardic and mainly in the increase in the frequency of the PND to microinjection of L-glutamate into the comNTS in the WHBP. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The nucleus tractus solitarius (NTS) is the site in the brain where afferents mediating different cardiovascular and respiratory reflexes establish their primary synapse. The commis-
sural subnucleus of the NTS (comNTS) encompasses a region that extends from the rostral edge of the area postrema to 1 mm caudal to the calamus scriptorius (CS; Drhuva et al., 1998). The chemoreceptor afferents have been reported to terminate predominantly in the region of the comNTS caudal
⁎ Corresponding author. Fax: +55 16 633 0017. E-mail address: [email protected] (B.H. Machado). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.105
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to the CS (0–0.5 mm caudal, 0–0.5 mm lateral and 0.3–0.5 deep with respect to the calamus scriptorius; Sapru, 1996, 2004). On the other hand, there is evidence that the baroreceptor and cardiopulmonary afferents terminate in a region rostral and lateral to chemoreceptor projection site (Vardhan et al., 1993; Chitravanshi et al., 1994; Chitravanshi and Sapru, 1995, 1996; Marchenko and Sapru, 2000). Although the excitatory amino acid (EAA) L-glutamate is considered the most important neurotransmitter released in the NTS by the afferents of cardiovascular reflexes (Talman et al., 1980; Talman, 1989; Vardhan et al., 1993; Zhang and Mifflin, 1993), the involvement of this EAA in the neurotransmission of the chemoreflex, for example, is controversial due to different experimental approaches (Vardhan et al., 1993; Zhang and Mifflin, 1993; Haibara et al., 1995, 1999; Machado and Bonagamba, 2005). In addition, cardiovascular responses to microinjection of L-glutamate into the comNTS may also vary in accordance with the experimental approach used. Microinjection of L-glutamate into the comNTS rostral and lateral to the CS of anesthetized or unanesthetized rats produces baroreflex-like responses (Leone and Gordon, 1989; Talman et al., 1980; Talman, 1989; Canesin et al., 2000),
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whereas L-glutamate microinjected into the comNTS at the CS level of unanesthetized rats elicits increase in arterial pressure and bradycardia, a pattern of cardiovascular adjustments similar to that produced by the chemoreflex activation (Machado and Bonagamba, 1992; Colombari et al., 1994, 1996; Machado, 2001, 2004). With respect to the respiratory effects of EAA receptor activation in the NTS, the data available are also controversial. Studies by Mizusawa et al. (1994) performed in unanesthetized rats showed that local microinjection of L-glutamate into the comNTS, caudal to the CS, increases ventilation by acting on NMDA receptors. In addition, studies by Bonham and McCrimmon (1990), performed in rats under urethane anesthesia, showed that the microinjection of DL-homocysteic acid into the NTS, from 0.8 mm rostral to 0.2 mm caudal to CS and from the midline to 2 mm lateral, inhibits phrenic nerve discharge by activation of receptors involved in the Hering–Breuer reflex. Besides, studies from Vitagliano et al. (1994), performed in rats under urethane anesthesia, showed that unilateral microinjection of L-glutamate or a metabotropic glutamate receptor agonist (trans-ACPD) into the NTS, 1.4 mm rostral and 0.5 lateral with respect to CS, produced apnea.
Fig. 1 – Effects of microinjection of L-glutamate into the comNTS on heart rate (HR), frequency of phrenic nerve discharge (PND) and thoracic sympathetic nerve activity (tSNA). Panel A illustrates a group of tracings from a representative WHBP showing the raw tracings of ECG and PND, the changes in HR, ∫ PND, ∫ tSNA and the raw tSNA in response to microinjection of L-glutamate (50 mM) into the comNTS. Panels B–D represent the changes in the heart rate ( ΔHR, bpm), in the frequency of the PND (Hz) and in the tSNA ( Δ%), respectively, in response to microinjection of increasing concentrations of L-glutamate into the comNTS (5, 25, 50, 250 and 500 mM in 20 nl; n = 9).
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Considering that the involvement of L-glutamate and its ionotropic and metabotropic glutamate receptors within the NTS on the cardiovascular and mainly on the respiratory neural mechanism are not completely understood, in the present study we investigated the autonomic and respiratory responses to microinjection of L-glutamate into the comNTS before and after local microinjection of ionotropic and metabotropic glutamate receptor antagonists. To reach these goals, we performed microinjections into the comNTS in the working heart–brainstem preparation (WHBP), which is characterized as an in situ unanesthetized preparation, and allow the simultaneous evaluation of the changes in the frequency of the phrenic nerve discharge, thoracic sympathetic nerve activity and heart rate.
2.
Results
2.1. Concentration–response curves to microinjection of L-glutamate into the comNTS Fig. 1A shows the typical changes in the HR, integrated PND, integrated tSNA and in the raw tSNA in response to microinjection of 50 mM of L-glutamate into the comNTS in the WHBP. Figs. 1B–D summarize the data from 9 WHBPs and show that increasing concentrations of L-glutamate (5, 25, 50, 250 and 500 mM) produced bradycardia (−9 ± 3, −29 ± 2, −64 ± 3, −103 ± 3 and −114 ± 3 bpm), reduction in the frequency of the PND in absolute values (0.29 ± 0.03, 0.20 ± 0.02, 0.09 ± 0.02, 0.03 ± 0.02 and 0.00 ± 0.00 Hz) and sympathoexcitation (0 ± 0.5%, 5 ± 1%, 10 ± 1%, 14 ± 1% and 22 ± 4%), respectively, in a concentration-dependent manner. Microinjection of the vehicle (saline) into the same site in the NTS produced negligible changes in the HR (−2 ± 2 bpm), frequency of PND (0.35 ± 0.021 Hz) and tSNA (1.1 ± 0.5%). The changes in the frequency of the PND in response to increasing concentrations of L-glutamate microinjected into the NTS on time domain are shown in Fig. 3A.
2.2. Changes in the HR, frequency of PND and tSNA in response to microinjection of L-glutamate before and after local microinjection of kynurenic acid (250 mM) into the comNTS Fig. 2 summarizes the changes in the frequency of the PND (panel A), HR (panel B) and tSNA (panel C) in response to microinjection of L-glutamate (50 mM) before and at 2, 10, 30 and 45 min after the antagonism of the ionotropic glutamate receptors with kynurenic acid (KYN, 250 mM) in the comNTS in 6 WHBPs. Control microinjection of the L-glutamate into the NTS produced a reduction in the frequency of the PND [from 0.36 ± 0.02 (dashed line) to 0.07 ± 0.03 Hz, panel A] before antagonism of ionotropic glutamate receptor with KYN. Microinjection of L-glutamate at 2 min after the local microinjection of KYN into the same site in the NTS produced an increase in the frequency of the PND compared to the baseline [from 0.36 ± 0.02 (dashed line) to 0.55 ± 0.02 Hz]. L-Glutamate microinjected at 10 min also produced increase in the frequency of the PND, when
Fig. 2 – Changes in the frequency of PND (Hz, panel A), in the heart rate ( ΔHR, bpm, panel B) and in the tSNA ( Δ%, panel C) in response to microinjection of L-glutamate (50 mM) before and at 2, 10, 30 and 45 min after the blockade of the ionotropic glutamate receptors with kynurenic acid (KYN, 250 mM) in the comNTS (n = 6). Dashed line indicates the baseline of the frequency of PND (0.36 ± 0.02 Hz). *Different when compared to the control microinjection of L-glutamate (P < 0.05).
compared to the baseline [from 0.36 ± 0.02 (dashed line) to 0.65 ± 0.02 Hz]. At 30 and 45 min after KYN, the pattern of responses elicited by L-glutamate was back to the control. Regarding the HR (panel B), the bradycardia produced by the microinjection of the L-glutamate into the comNTS was significantly reduced at 2 (from −83 ± 11 to −23 ± 8 bpm) and 10 min (from −83 ± 11 to −26 ± 5 bpm) after the antagonism of the ionotropic glutamate receptors with KYN microinjected into the same site in the NTS. The control increase in the tSNA (+7.5 ± 0.6%, panel C) produced by the microinjection of L-glutamate into the comNTS was almost abolished at 2 and 10 min after the microinjection of KYN into the same
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site in the NTS. The bradycardic response and the increase in tSNA in response to microinjection of Lglutamate into the comNTS were back to control levels 30 min after the microinjection of KYN. The unilateral microinjection of KYN produced no changes on the baseline of the HR, PND and tSNA. The changes in the frequency of the PND in response to L-glutamate microinjected into the NTS before and after KYN on time domain are shown in Fig. 3B.
2.3. Concentration–response curves to microinjection of trans-ACPD into the comNTS Fig. 4A shows the typical tracings of one WHBP in which microinjection of trans-ACPD (2.5 mM) was performed into the comNTS and the changes in HR, in the frequency of the PND and in the tSNA (integrated and raw) were evaluated. Panels B– D show that increasing concentrations of trans-ACPD (0.5, 1.0, 2.5, 5.0 and 10 mM) produced bradycardia (−4 ± 2, −35 ± 4, −46 ± 6, −63 ± 10 and −102 ± 9 bpm) and increase in the frequency of the PND (0.44 ± 0.02, 0.53 ± 0.04, 0.60 ± 0.02, 0.67 ± 0.03 and 0.74 ± 0.03 Hz) in a concentration-dependent manner and no major changes in the tSNA (0.22 ± 0.84%, 0.66 ± 0.32%, −1.30 ± 0.40%, 0.70 ± 0.34% and −0.62 ± 1.76%). Microinjection of the vehicle (saline) at the same level of the NTS produced negligible changes on the HR (−4 ± 2 bpm), frequency of the PND (0.37 ± 0.02 Hz) and tSNA (−1.1 ± 0.6%). The changes in the frequency of the PND in response to increasing concentrations of trans-ACPD microinjected into the NTS on time domain are shown in Fig. 6A.
2.4. Changes in the HR and frequency of the PND in response to microinjection of trans-ACPD before and after the antagonism of metabotropic glutamate receptors (MCPG, 100 mM) into the comNTS Fig. 5 summarizes the changes in the frequency of the PND (panel A) and HR (panel B) in response to microinjection of
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trans-ACPD (2.5 mM) before and at 2, 10, 30 and 45 min after the antagonism of metabotropic glutamate receptors with MCPG (100 mM) into the comNTS in the WHBP (n = 5). Microinjection of trans-ACPD (2.5 mM) produced an increase in the frequency of the PND [from 0.37 ± 0.03 (dashed line) to 0.68 ± 0.02 Hz]. The increase in the frequency of the PND produced by trans-ACPD microinjected into the comNTS was abolished at 2 [from 0.37 ± 0.02 (dashed line) to 0.36 ± 0.02 Hz] and 10 min [from 0.37 ± 0.02 (dashed line) to 0.34 ± 0.02 Hz] after antagonism of metabotropic glutamate receptors with MCPG at the same site in the NTS. At 45 min, the increase in the frequency of the PND was back to the control values. Regarding the bradycardic response produced by microinjection of trans-ACPD into the comNTS, it was significantly reduced at 2 (from −55 ± 2 to −7 ± 1 bpm), 10 (from −55 ± 2 to −5 ± 1 bpm) and 30 min (from −55 ± 2 to −25 ± 4 bpm) after the blockade of metabotropic glutamate receptors with MCPG, and it was back to the control values 45 min after the antagonism. The unilateral microinjection of MCPG produced no changes on the baseline of the HR, PND and tSNA. The changes in the frequency of the PND in response to trans-ACPD microinjected into the comNTS before and after MCPG on time domain are shown in Fig. 6B.
2.5. Changes in the HR and frequency of the PND in response to microinjection of L-glutamate before and after the simultaneous blockade of ionotropic (KYN, 250 mM) and metabotropic (MCPG, 100 mM) glutamate receptors into the comNTS Fig. 7 shows the changes in the frequency of the PND (panel A) and HR (panel B) in response to microinjection of L-glutamate (50 mM) before (control) and at 2, 10, 30 and 45 min after microinjection of both ionotropic (KYN, 250 mM) and metabotropic (MCPG, 100 mM) glutamate receptors into the commissural subnucleus of the NTS in the WHBP. Microinjection of L-glutamate produced a reduction in the frequency
Fig. 3 – (A) Changes in the frequency of the PND in the 50 s subsequent to microinjections of increasing concentrations of L-glutamate into the comNTS. (B) Changes in the frequency of the PND in the 50 s subsequent to microinjection of L-glutamate (50 mM) into the comNTS before and after KYN (250 mM). *Different when compared to saline (A) or control microinjection of L-glutamate (B) (P < 0.05).
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Fig. 4 – Effects of microinjection of trans-ACPD microinjected into the comNTS on heart rate (HR), frequency of phrenic nerve discharge (PND) and thoracic sympathetic nerve activity (tSNA). Panel A illustrates a group of tracings from a representative WHBP showing the raw tracings of ECG and PND, the changes in HR, ∫ PND, ∫ tSNA and the raw tSNA in response to microinjection of trans-ACPD (2.5 mM) into the comNTS. Panels B–D represent the changes in the heart rate ( ΔHR, bpm), in the frequency of the PND (Hz) and in the tSNA ( Δ%), respectively, in response to microinjection of increasing concentrations of trans-ACPD into the comNTS (0.5, 1, 2.5, 5 and 10 mM in 20 nl; n = 7).
of the PND (from 0.39 ± 0.03 to 0.07 ± 0.03 Hz), and at 2 and 10 min after the double blockade, the changes in the frequency of PND were almost abolished (from 0.47 ± 0.03 to 0.58 ± 0.04 Hz and from 0.49 ± 0.03 to 0.50 ± 0.06 Hz, respectively). At 30 and 45 min after the double blockade (KYN + MCPG), the reduction in the frequency of the PND in response to microinjection of L-glutamate into the comNTS was back to the control. With respect to the bradycardic response produced by microinjection of L-glutamate into the comNTS, it was also significantly reduced at 2 (from −76 ± 7 to −19 ± 5 bpm) and 10 min (from −76 ± 7 to −18 ± 3 bpm) after the double blockade. This pattern of attenuation of the magnitude of the bradycardic response was similar to the attenuation produced by the blockade of ionotropic receptors with KYN (Fig. 2B). The unilateral microinjection of KYN + MCPG produced no changes on the baseline of the HR, PND and tSNA. The changes in frequency of PND in response to L-glutamate microinjected into the NTS before
and after the double blockade (KYN + MCPG) on time domain are shown in Fig. 8.
2.6.
Histology
Fig. 9A presents a photomicrography of a coronal section of the brainstem of one WHBP representative of the groups showing the track of the micropipette in the direction of the comNTS and also the center of the microinjection. Fig. 9B is a diagram of a coronal section of the medulla showing the sites of microinjections of L-glutamate into the comNTS of a group of 9 WHBPs.
3.
Discussion
The experimental approach of microinjection into the NTS of awake rats has the advantage of being performed in an
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Fig. 5 – (A) Changes in the frequency of PND (Hz) in response to microinjection of trans-ACPD (2.5 mM) before and at 2, 10, 30 and 45 min after the blockade of metabotropic glutamate receptors with MCPG (100 mM) into the comNTS (n = 5). (B) Changes in the heart rate ( ΔHR) in response to the same microinjection. Dashed line indicates the baseline of the frequency of PND (0.37 ± 0.03 Hz). *Different when compared to the control microinjection of trans-ACPD (P < 0.05).
anesthetic-free neural environment, whereas the experiments performed in anesthetized rats have the advantage of the use of glass micropipettes, which allows microinjections of volumes smaller than those obtained with the needles used in awake rats. It is important to consider that under anesthesia, the magnitude of the cardiovascular and respiratory responses to microinjections into the NTS or to activation of different reflexes may vary (Machado and Bonagamba, 1992). In the present study, we used a decerebrated artificially perfused preparation (Paton, 1996), which presents the following advantages: (a) cardiovascular reflexes and eupneic respiratory pattern are preserved; (b) activities from the sympathetic and phrenic nerves can be recorded simultaneously; (c) small volumes of microinjections can be performed into the NTS under direct microscopic observation scope; and (d) the effects of anesthetics are prevented. On the other hand, this preparation also presents some disadvantage, such as the loss of descending influences from the midbrain and higher centers due to the decerebration.
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In the present study, performed in an in situ unanesthetized preparation, we observed several important findings with respect to the autonomic and respiratory responses related to activation of ionotropic and metabotropic glutamate receptors in the comNTS caudal to the CS. Among these findings, we highlight the following: (a) in the WHBP, the microinjection of L-glutamate (50 mM, 20 nl) into the comNTS produced bradycardia, increase in thoracic sympathetic nerve activity and reduction in the frequency of the PND; (b) both bradycardia and increase in tSNA in response to L-glutamate were attenuated by previous microinjection of kynurenic acid (250 mM, 20 nl) into the same site in the NTS; (c) the reduction in the frequency of the PND produced by microinjection of L-glutamate into the comNTS was not only blocked by kynurenic acid but also reversed to an increase in the frequency of the PND; (d) the increase in the frequency of the PND produced by L-glutamate microinjected into the comNTS after blockade of the ionotropic glutamate receptors was abolished after microinjection of MCPG (100 mM, 20 nl) into the same site in the NTS, indicating that metabotropic receptors are involved in the observed increase in the frequency of the PND; and (e) the microinjection of increasing doses of a metabotropic glutamate receptor agonist (trans-ACPD) produced increase in the frequency of the PND in a concentration-dependent manner, supporting the concept that metabotropic glutamate receptors are involved in the in the increase in the frequency of the PND. Our data support the conclusion that microinjection of Lglutamate into the comNTS activates mainly ionotropic glutamate receptors. First, the response of L-glutamate was significantly attenuated by previous NTS administration of kynurenic acid, a selective ionotropic glutamate receptor antagonist, a pattern of response similar to those observed in previous studies (Bertolino et al., 1989; Leone and Gordon, 1989; Andresen and Kunze, 1994). Second, several studies demonstrated that microinjection of selective ionotropic glutamate receptor agonists into the NTS produce responses similar to those produced by L-glutamate, and also that these responses were inhibited by kynurenic acid (Kubo and Kihara, 1991; Leone and Gordon, 1989; Le Galloudec et al., 1989; Talman, 1989; Almado and Machado, 2005). Our data show that L-glutamate acting on ionotropic glutamate receptors in the comNTS caudal to CS produces bradycardia, increase in tSNA and reduction in the frequency of the PND. The autonomic responses elicited by microinjection of Lglutamate into the comNTS in the present study (sympathoexcitation and bradycardia) were similar to the pattern of responses elicited by chemoreflex activation in the WHBP (Antunes et al., 2005), nevertheless, with less intensity. In addition, these autonomic responses were attenuated by KYN microinjected into the same site in the NTS indicating a role for the ionotropic glutamate receptors. Although these responses are chemoreflex-like, these data do not allow us to suggest that L-glutamate is exclusively acting on neurons involved in the processing of the chemoreflex pathways at the comNTS level. In fact, the sites shown in Fig. 9 suggest that medial portions of the regions from
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Fig. 6 – (A) Changes in the frequency of the PND in the 50 s subsequent to microinjections of increasing concentrations of trans-ACPD into the comNTS. (B) Changes in the frequency of the PND in the 50 s subsequent to microinjection of trans-ACPD (2.5 mM) into the comNTS before and after MCPG (100 mM). *Different when compared to saline (A) or control microinjection of trans-ACPD (B) (P < 0.05).
which baroreceptor-like responses are elicited by L-glutamate may have also contributed to the observed responses in this study. For this purpose, further studies using ionotropic glutamate receptors antagonists against the chemoreflex activation itself are required to evaluate this interesting possibility. The reduction in the frequency of the PND produced by microinjection of L-glutamate into the comNTS may be related to the activation of neurons involved in the Hering– Breuer reflex pathways identified as “pump cells” (Bonham and McCrimmon, 1990; Bonham et al., 1993; Berger et al., 1995). The activation of these neurons produces a reduction in the frequency of the PND that seems to be mediated by ionotropic glutamate receptors (Bonham et al., 1993; Rybak et al., 2004). The involvement of ionotropic glutamate receptors in the reduction in the frequency of the PND produced by microinjection of L-glutamate into the comNTS is strongly supported by the fact that the microinjection of kynurenic acid into the same site in the NTS blocked this response. In addition to the activation of ionotropic receptors, L-glutamate also activates metabotropic receptors in the comNTS, producing an increase in the frequency of the PND as observed when L-glutamate was microinjected after blockade of ionotropic glutamate receptors with KYN. This increase in the frequency of the PND was blocked when metabotropic glutamate receptor antagonist (MCPG) was combined with KYN. The involvement of metabotropic receptors in the increase in the frequency of the PND to microinjection of L-glutamate after the blockade of ionotropic receptors with kynurenic acid is also supported by the data from the experimental protocol using increasing doses of trans-ACPD, which clearly demonstrated that the activation of metabotropic receptors produced a dose-dependent increase in the frequency of the PND.
In conclusion, microinjection of L-glutamate into the comNTS caudal to CS activates ionotropic receptors producing sympathoexcitation, whereas the bradycardic response to L-glutamate seems to be mediated by both ionotropic and metabotropic receptors activation. The most exciting finding of the present study is related to the changes in the frequency of the PND because L-glutamate activating ionotropic receptors produces reduction in the frequency of the PND whereas the activation of metabotropic receptors produces increase in the frequency of the PND. Considering that our goal was to characterize the autonomic and respiratory response to microinjection of L-glutamate into the comNTS caudal to CS, the data obtained in the present study open an interesting field to explore the involvement of L-glutamate and its ionotropic and metabotropic receptors in the neurotransmission and neuromodulation of the autonomic and respiratory components of the cardiovascular reflexes in the commissural subnucleus of the NTS.
4.
Experimental procedure
4.1.
General surgical procedures
The experiments were performed in an in situ unanesthetized decerebrated WHBP as described previously by Paton (1996). Male Wistar rats (70–90 g) were anesthetized deeply with halothane and the level of anesthesia was assessed by absence of response to a noxious pinch of either the paw or the tail. Following subdiaphragmatic transection, the rostral half of the animal was submerged in cooled artificial cerebrospinal fluid (ACSF) carbogen gassed (95% O2 and 5% CO2), decerebrated at the precollicular level and skinned, the
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Medicine approved December Brazilian (COBEA).
4.2.
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of Ribeirão Preto, University of São Paulo, all procedures during the ordinary meeting on 1, 2003, according to recommendations of the Association for Laboratory Animal Science
Recordings of electrocardiogram and nerve activities
Left phrenic nerve activity was recorded from its central end using a glass suction electrode held in a micromanipulator. Rhythmic ramping phrenic nerve discharge (PND) gave a continuous physiological index of the preparation viability. The electrocardiogram (ECG) was visible on the phrenic nerve recording, which allowed us evaluate the heart rate (HR) by using a low-pass filter (Figs. 1A and 4A). Sympathetic nerve activity was recorded from the thoracic sympathetic chain (tSNA) at the level of T5–T10 using a second glass suction bipolar electrode. Signals were AC amplified, band-pass filtered (8 Hz to 3 kHz) and displayed on a computer using the software Spike 2 (Cambridge Electronic Design, Cambridge, England).
4.3.
Fig. 7 – (A) Changes in the frequency of PND (Hz) in response to microinjection of L-glutamate (50 mM) before and at 2, 10, 30 and 45 min after the double blockade of ionotropic and metabotropic glutamate receptors with kynurenic acid (KYN, 250 mM) combined with MCPG (100 mM) into the comNTS (n = 6). (B) Changes in the heart rate ( ΔHR) in response to the same microinjection. Dashed line indicates the baseline of the frequency of PND (0.47 ± 0.03 Hz at 2 min after the blockade). *Different when compared to the control microinjection of L-glutamate (P < 0.05).
Microinjections into the NTS
The calamus scriptorius (CS) was used as a landmark for microinjections into the comNTS. Drugs were applied unilaterally via a three-barreled micropipette (tip diameter 20–30 μm). The tip of the micropipette was driven into the medulla to a depth of 0.3–0.4 mm ventral to the dorsal surface, 0.2–0.4 mm caudal relative to CS and between 0.25 and 0.35 mm from midline. The injected volume for all drugs (20 nl) was determined by meniscus calibration in the eyepiece of the microscopy. At the end of the experiments, the brain was removed, fixed and histological procedures were performed in order to verify the micropipette track and the center of microinjections in the comNTS.
4.4.
Agonists and antagonists microinjected into the NTS
L-Glutamate,
descending aorta was then isolated, the heart exposed by removal of the left ribs and the lungs removed. The dorsal surface of the brainstem was exposed by removal of the occipital bone and cerebellum. The WHBP was moved to a recording chamber, then the descending aorta was cannulated and perfused retrogradely with ACSF (in mM): NaCl, 125; NaHCO3, 24; KCl, 5; CaCl2, 2.5; MgSO4, 1.25; KH2PO4, 1.25; dextrose, 10; oncotic agent (Ficoll® 70, 1.25%; Sigma, St. Louis, MO, USA) using a roller pump (Watson-Marlow 502s, Falmouth, Cornwall, UK) via double lumen cannula. A neuromuscular blocker (vecuronium bromide, 0.04 mg/ ml; Norcuron Organon Teknika, Brazil) was used for stopping the spontaneous chest wall respiratory movements. Perfusion pressure was maintained in a narrow range (from 50 to 70 mm Hg) by adjusting flow rate of the perfusion pump. The perfusate was gassed with carbogen continuously, warmed to 32 °C and filtered using a nylon mesh (pore size: 25 μm; Millipore, Ireland). The Ethical Committee for Animal Experimentation of the School of
kynurenic acid (KYN), trans-1-amino-1,3-cyclopentanediocarboxylic acid (trans-ACPD) and (±)-α-methyl-4carboxyphenylglycine (MCPG) were obtained from Sigma (St. Louis, MO, USA). Drugs were dissolved in saline (NaCl 0.9%) and adjusted to pH 7.4 by adding NaHCO3. Concentrations are expressed as the free base of each drug. The concentration of 250 mM of KYN was the concentration that attenuated all the responses to L-glutamate microinjection into the NTS (sympathoexcitation, bradycardia and reduction in the frequency of PND) without affecting the baseline of the parameters evaluated. In a preliminary study, we tested two other concentrations of KYN (50 and 100 mM). These concentrations produced no changes in the sympathoexcitation and in the reduction in the phrenic nerve discharge elicited by microinjection of Lglutamate into the NTS [50 mM: tSNA (+13 ± 3 vs. +11 ± 4%) and PND (from 0.36 ± 0.02 to 0.11 ± 0.02 Hz vs. 0.35 ± 0.03 to 0.33 ± 0.02 Hz); and 100 mM: tSNA (+12 ± 2 vs. +13 ± 3%) and PND (from 0.35 ± 0.03 to 0.09 ± 0.04 Hz vs. 0.36 ± 0.04 to 0.35 ± 0.03 Hz)]. The
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n = 4). The concentration of MCPG (100 mM) was used in accordance with previous studies from our laboratory in which this concentration was effective in blocking the responses elicited by microinjection of trans-ACPD in the NTS (Antunes and Machado, 2003).
4.5.
Fig. 8 – Changes in the frequency of the PND in the 50 s subsequent to microinjection of L-glutamate (50 mM) into the comNTS before and after KYN (250 mM) combined with MCPG (100 mM). *Different when compared to the control microinjection of L-glutamate (P < 0.05). concentration of 100 mM of KYN produced only a significant reduction in the bradycardia produced by the microinjection of L-glutamate (−72 ± 6 vs. −42 ± 7 Δbpm,
Data analysis
All data were analyzed off-line using Spike2 software with custom-written scripts. Baseline and peak responses in HR and tSNA were measured. Frequency of bursts of the PND was measured in a time-dependent way (counted each 10 s starting 40 s before the microinjection and ending 50 s after microinjection). Besides, we evaluated the peak of the response (frequency of PND) after each microinjection. The rectified and integrated signal of the tSNA (100 ms time constant) was measured for a period covering 10 s before and 10s after each microinjection as previously described (Braga and Machado, 2006). The data from tSNA were expressed as percentage of the control. The significance of effects was assessed by twoway ANOVA followed by Tukey's post hoc test (P < 0.05) to evaluate the time course responses in the frequency of PND and one-way ANOVA followed by Tukey's post hoc test (Pstandard error (SE) and n is the number of preparations.
Fig. 9 – (A) Photomicrography of a transverse section of the brainstem of one rat, representative of the group, showing the unilateral microinjection site in the commissural subnucleus of the NTS. The arrow indicates the center of the microinjection in the NTS. Scale bar corresponds to 500 μm. (B) Diagram of a coronal section of the medulla showing the site of microinjections of L-glutamate into the comNTS caudal to CS (n = 9). CC, central canal; 10 dorsal motor nucleus, 12 hypoglossal nucleus.
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Acknowledgments The authors thank Prof. Julian F.R. Paton, University of Bristol, UK, and Prof. Jeffrey T. Potts, University of Missouri, EUA, for their intellectual and material support to the development of the WHBP in our laboratory. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2001/11190-8 and 2004/03285-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ, 472704/ 2004-4).
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Almado, C.E., Machado, B.H., 2005. Respiratory and autonomic responses to microinjection of NMDA and AMPA into the commissural subnucleus of the NTS of awake rats. Brain Res. 1063 (1), 59–68. Andresen, M.C., Kunze, D.L., 1994. Nucleus tractus solitarius— Gateway to neural circulatory control. Annu. Rev. Physiol. 56, 93–116. Antunes, V.R., Machado, B.H., 2003. Antagonism of glutamatergic metabotropic receptors in the NTS of awake rats does not affect the gain of the baroreflex. Auton. Neurosc. 103, 65–71. Antunes, V.R., Braga, V.A., Machado, B.H., 2005. Autonomic and respiratory responses to microinjection of ATP into the intermediate or caudal nucleus tractus solitarius in the working heart–brainstem preparation of the rat. Clin. Exp. Pharmacol. Physiol. 32, 467–472. Berger, I., Gillis, R.A., Vitagliano, S., Panico, W.H., Magee, S., Kelly, M., Norman, W.P., McManigle, J.E., Taveira DaSilva, A.M., 1995. NMDA receptors are involved at the ventrolateral nucleus tractus solitarii for termination of inspiration. Eur. J. Pharmacol. 277 (2–3), 195–208. Bertolino, M., Vicini, S., Costa, E., 1989. Kynurenic acid inhibits the activation of kainic and N-methyl-D-aspartic acid-sensitive ionotropic receptors by a different mechanism. Neuropharmacology 28, 543–547. Bonham, A.C., McCrimmon, D.R., 1990. Neurons in a discrete region of the nucleus tractus solitarius are required for the Breuer–Hering reflex in rat. J. Physiol. 427, 261–280. Bonham, A.C., Coles, S.K., McCrimmon, D.R., 1993. Pulmonary stretch receptor afferents activate excitatory amino acid receptors in the nucleus tractus solitarii in rats. J. Physiol. 464, 245–725. Braga, V.A., Machado, B.H., 2006. Chemoreflex sympathoexcitation was not altered by the antagonism of glutamate receptors in the commissural NTS in the working heart–brainstem preparation of rat. Exp. Physiol. 91 (3), 551–559. Canesin, R.O., Bonagamba, L.G.H., Machado, B.H., 2000. 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 Res. 852, 68–75. Chitravanshi, V.C., Sapru, H.N., 1995. Chemoreceptor-sensitive neurons in commissural subnucleus of nucleus tractus solitarius of the rat. Am. J. Physiol. 268, 851–858. Chitravanshi, V.C., Kachroo, A., Sapru, H.N., 1994. A midline area in the nucleus commissuralis of NTS mediates the phrenic nerve responses to carotid chemoreceptor stimulation. Brain Res. 662, 127–132. Colombari, E., Bonagamba, L.G.H., Machado, B.H., 1994.
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Mechanisms of pressor and bradycardic responses to microinjected into the NTS of conscious rats. Am. J. Physiol. 266, R730–R738. Colombari, E., Menani, J.V., Talman, W.T., 1996. Commissural NTS contributes to pressor responses to glutamate injected into medial NTS of awake rats. Am. J. Physiol. Reg. Integ. Comp. Physiol. 270 (39), R1220–R1225. Drhuva, A., Bhatnagar, T., Sapru, H.N., 1998. Cardiovascular responses to microinjections of glutamate into the nucleus tractus solitarii of unanaesthetized supracollicular decerebrated rats. Brain Res. 801, 88–100. Haibara, A.S., Colombari, E., Chianca Jr., D.A., Bonagamba, L.G.H., Machado, B.H., 1995. NMDA receptors in the NTS are involved in bradycardic but not in pressor response of chemoreflex. Am. J. Physiol.: Heart Circ. Physiol. 269, H1421–H1427. Haibara, A.S., Bonagamba, L.G.H., Machado, B.H., 1999. Sympathoexcitatory neurotransmission of the chemoreflex in the NTS of awake rats. Am. J. Physiol. Reg. Integr. Physiol. 276, R69–R80. Kubo, T., Kihara, M., 1991. Unilateral blockade of excitatory amino acid receptors in the nucleus tractus solitarii produces an inhibition of baroreflexes in rats Naunyn-Schmiedeberg's Arch. Pharmacol. 343, 317–322. Le Galloudec, E., Merahi, N., Laguzzi, R., 1989. Cardiovascular changes induced by the local application of glutamate-related drugs in the rat nucleus tractus solitarii. Brain Res. 503, 322–325. Leone, C., Gordon, F.J., 1989. Is L-glutamate a neurotransmitter of baroreceptor information in the nucleus of the tractus solitarius? J. Pharmacol. Exp. Ther. 250, 953–962. Machado, B.H., 2001. Neurotransmission of the cardiovascular reflexes in the nucleus tractus solitarii of awake rats. An. New York Acad. Sci. 940, 179–196. Machado, B.H., 2004. Chemoreflex and sympathoexcitation. In: Dun, N.J., Machado, B.H., Pilowsky, P.M. (Eds.), Neural Mechanisms of Cardiovascular Regulation. Kluwer Academic Publishers, Norwell, MA. 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. Machado, B.H., Bonagamba, L.G.H, 2005. Antagonism of glutamate receptors in the intermediate and caudal NTS of awake rats produced no changes in the hypertensive response to chemoreflex activation. Auton. Neurosc. 117, 25–32. Marchenko, V., Sapru, H.N., 2000. Different patterns of respiratory and cardiovascular responses elicited by chemical stimulation of dorsal medulla in the rat. Brain Res. 857, 99–109. Mizusawa, A., Ogawa, H., Kikuchi, Y., Hida, W., Kurosawa, H., Okabe, S., Takishima, T., Shirato, K., 1994. In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia. J. Physiol. 478:1, 55–65. Paton, J.F.R., 1996. A working heart–brainstem preparation of the mouse. J. Neurosci. Methods 65, 63–68. Rybak, I.A., Shevtsova, N.A., Paton, J.F.R., Dick, T.E., St-John, W.M., Morschel, M., Dutschmann, M., 2004. Modeling the ponto-medullary respiratory network. Respir. Physiol. Neurobiol. 143 (2–3), 307–319. Sapru, H.N., 1996. Carotid chemoreflex. Neural pathways and transmitters. Adv. Exp. Med. Biol. 410, 357–364. Sapru, H.N., 2004. Neurotransmitters in the nucleus tractus solitarius mediating cardiovascular function. In: Dun, N.J., Machado, B.H., Pilowsky, P.M. (Eds.), Neural Mechanisms of Cardiovascular Regulation. Kluwer Academic Publishers, Norwell, MA. Talman, W.T., 1989. Kynurenic acid microinjected into the nucleus L-glutamate
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tractus solitarius of rat blocks the arterial baroreflex but not responses to glutamate. Neurosci. Lett. 102, 247–252. Talman, W.T., Perrone, M.H., Reis, D.J., 1980. Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers. Science 209, 813–815. 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.
Vitagliano, S., Berrino, L., Pizzirusso, A., D'amico, M., Caderaro, V., Maione, S., Rossi, F., 1994. Metabotropic glutamate receptors are involved in the control of breathing at the medulla oblongata level of anaesthetized rats. Neuropharmacology 37:7, 859–864. Zhang, W., Mifflin, S.W., 1993. Excitatory amino acid receptors within NTS mediate arterial chemoreceptor reflexes in rats. Am. J. Physiol. 265, H770–H773.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Autonomic and respiratory responses to microinjection of L-glutamate into the commissural subnucleus of the NTS in the working heart–brainstem preparation of the rat Valdir A. Braga, Vagner R. Antunes, Benedito H. Machado⁎ Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Changes in heart rate (HR), thoracic sympathetic nerve activity (tSNA) and frequency of
Accepted 20 March 2006
phrenic nerve discharge (PND) in response to microinjection of L-glutamate before and
Available online 16 May 2006
after local microinjection of ionotropic or metabotropic glutamate receptors antagonists into the commissural subnucleus of the NTS (comNTS) were investigated. The
Keywords:
experiments were performed in an in situ unanesthetized decerebrated working
L-glutamate
heart–brainstem preparation (WHBP), and the main findings were as follows: (a)
Ionotropic receptor
microinjection of increasing concentrations of L-glutamate (5, 25, 50, 250 and 500 mM)
Metabotropic receptor
into the comNTS produced bradycardia, increase in tSNA and reduction in the
Phrenic nerve discharge
frequency of the PND in a concentration-dependent manner; (b) both bradycardia and
Sympathetic nerve activity
increase in tSNA were almost abolished by kynurenic acid (KYN, 250 mM, a nonselective
Heart rate
ionotropic glutamate receptor antagonist); (c) the reduction in the frequency of the PND was reversed to an increase in the frequency of the PND after KYN and this increase was blocked by the sequential microinjection of MCPG (100 mM, a nonselective metabotropic glutamate receptor antagonist); and (d) microinjection of increasing concentrations of trans-ACPD (0.5, 1.0, 2.5, 5.0 and 10 mM, a metabotropic glutamate receptor agonist), elicited bradycardia and increase in the frequency of the PND in a concentration-dependent manner, which were blocked by MCPG. Taken together, these data indicate that
L-glutamate
and its ionotropic receptors are involved in the
sympathoexcitatory, bradycardic and reduction in the frequency of the PND responses whereas/although its metabotropic receptors are involved in the bradycardic and mainly in the increase in the frequency of the PND to microinjection of L-glutamate into the comNTS in the WHBP. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The nucleus tractus solitarius (NTS) is the site in the brain where afferents mediating different cardiovascular and respiratory reflexes establish their primary synapse. The commis-
sural subnucleus of the NTS (comNTS) encompasses a region that extends from the rostral edge of the area postrema to 1 mm caudal to the calamus scriptorius (CS; Drhuva et al., 1998). The chemoreceptor afferents have been reported to terminate predominantly in the region of the comNTS caudal
⁎ Corresponding author. Fax: +55 16 633 0017. E-mail address: [email protected] (B.H. Machado). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.105
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to the CS (0–0.5 mm caudal, 0–0.5 mm lateral and 0.3–0.5 deep with respect to the calamus scriptorius; Sapru, 1996, 2004). On the other hand, there is evidence that the baroreceptor and cardiopulmonary afferents terminate in a region rostral and lateral to chemoreceptor projection site (Vardhan et al., 1993; Chitravanshi et al., 1994; Chitravanshi and Sapru, 1995, 1996; Marchenko and Sapru, 2000). Although the excitatory amino acid (EAA) L-glutamate is considered the most important neurotransmitter released in the NTS by the afferents of cardiovascular reflexes (Talman et al., 1980; Talman, 1989; Vardhan et al., 1993; Zhang and Mifflin, 1993), the involvement of this EAA in the neurotransmission of the chemoreflex, for example, is controversial due to different experimental approaches (Vardhan et al., 1993; Zhang and Mifflin, 1993; Haibara et al., 1995, 1999; Machado and Bonagamba, 2005). In addition, cardiovascular responses to microinjection of L-glutamate into the comNTS may also vary in accordance with the experimental approach used. Microinjection of L-glutamate into the comNTS rostral and lateral to the CS of anesthetized or unanesthetized rats produces baroreflex-like responses (Leone and Gordon, 1989; Talman et al., 1980; Talman, 1989; Canesin et al., 2000),
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whereas L-glutamate microinjected into the comNTS at the CS level of unanesthetized rats elicits increase in arterial pressure and bradycardia, a pattern of cardiovascular adjustments similar to that produced by the chemoreflex activation (Machado and Bonagamba, 1992; Colombari et al., 1994, 1996; Machado, 2001, 2004). With respect to the respiratory effects of EAA receptor activation in the NTS, the data available are also controversial. Studies by Mizusawa et al. (1994) performed in unanesthetized rats showed that local microinjection of L-glutamate into the comNTS, caudal to the CS, increases ventilation by acting on NMDA receptors. In addition, studies by Bonham and McCrimmon (1990), performed in rats under urethane anesthesia, showed that the microinjection of DL-homocysteic acid into the NTS, from 0.8 mm rostral to 0.2 mm caudal to CS and from the midline to 2 mm lateral, inhibits phrenic nerve discharge by activation of receptors involved in the Hering–Breuer reflex. Besides, studies from Vitagliano et al. (1994), performed in rats under urethane anesthesia, showed that unilateral microinjection of L-glutamate or a metabotropic glutamate receptor agonist (trans-ACPD) into the NTS, 1.4 mm rostral and 0.5 lateral with respect to CS, produced apnea.
Fig. 1 – Effects of microinjection of L-glutamate into the comNTS on heart rate (HR), frequency of phrenic nerve discharge (PND) and thoracic sympathetic nerve activity (tSNA). Panel A illustrates a group of tracings from a representative WHBP showing the raw tracings of ECG and PND, the changes in HR, ∫ PND, ∫ tSNA and the raw tSNA in response to microinjection of L-glutamate (50 mM) into the comNTS. Panels B–D represent the changes in the heart rate ( ΔHR, bpm), in the frequency of the PND (Hz) and in the tSNA ( Δ%), respectively, in response to microinjection of increasing concentrations of L-glutamate into the comNTS (5, 25, 50, 250 and 500 mM in 20 nl; n = 9).
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Considering that the involvement of L-glutamate and its ionotropic and metabotropic glutamate receptors within the NTS on the cardiovascular and mainly on the respiratory neural mechanism are not completely understood, in the present study we investigated the autonomic and respiratory responses to microinjection of L-glutamate into the comNTS before and after local microinjection of ionotropic and metabotropic glutamate receptor antagonists. To reach these goals, we performed microinjections into the comNTS in the working heart–brainstem preparation (WHBP), which is characterized as an in situ unanesthetized preparation, and allow the simultaneous evaluation of the changes in the frequency of the phrenic nerve discharge, thoracic sympathetic nerve activity and heart rate.
2.
Results
2.1. Concentration–response curves to microinjection of L-glutamate into the comNTS Fig. 1A shows the typical changes in the HR, integrated PND, integrated tSNA and in the raw tSNA in response to microinjection of 50 mM of L-glutamate into the comNTS in the WHBP. Figs. 1B–D summarize the data from 9 WHBPs and show that increasing concentrations of L-glutamate (5, 25, 50, 250 and 500 mM) produced bradycardia (−9 ± 3, −29 ± 2, −64 ± 3, −103 ± 3 and −114 ± 3 bpm), reduction in the frequency of the PND in absolute values (0.29 ± 0.03, 0.20 ± 0.02, 0.09 ± 0.02, 0.03 ± 0.02 and 0.00 ± 0.00 Hz) and sympathoexcitation (0 ± 0.5%, 5 ± 1%, 10 ± 1%, 14 ± 1% and 22 ± 4%), respectively, in a concentration-dependent manner. Microinjection of the vehicle (saline) into the same site in the NTS produced negligible changes in the HR (−2 ± 2 bpm), frequency of PND (0.35 ± 0.021 Hz) and tSNA (1.1 ± 0.5%). The changes in the frequency of the PND in response to increasing concentrations of L-glutamate microinjected into the NTS on time domain are shown in Fig. 3A.
2.2. Changes in the HR, frequency of PND and tSNA in response to microinjection of L-glutamate before and after local microinjection of kynurenic acid (250 mM) into the comNTS Fig. 2 summarizes the changes in the frequency of the PND (panel A), HR (panel B) and tSNA (panel C) in response to microinjection of L-glutamate (50 mM) before and at 2, 10, 30 and 45 min after the antagonism of the ionotropic glutamate receptors with kynurenic acid (KYN, 250 mM) in the comNTS in 6 WHBPs. Control microinjection of the L-glutamate into the NTS produced a reduction in the frequency of the PND [from 0.36 ± 0.02 (dashed line) to 0.07 ± 0.03 Hz, panel A] before antagonism of ionotropic glutamate receptor with KYN. Microinjection of L-glutamate at 2 min after the local microinjection of KYN into the same site in the NTS produced an increase in the frequency of the PND compared to the baseline [from 0.36 ± 0.02 (dashed line) to 0.55 ± 0.02 Hz]. L-Glutamate microinjected at 10 min also produced increase in the frequency of the PND, when
Fig. 2 – Changes in the frequency of PND (Hz, panel A), in the heart rate ( ΔHR, bpm, panel B) and in the tSNA ( Δ%, panel C) in response to microinjection of L-glutamate (50 mM) before and at 2, 10, 30 and 45 min after the blockade of the ionotropic glutamate receptors with kynurenic acid (KYN, 250 mM) in the comNTS (n = 6). Dashed line indicates the baseline of the frequency of PND (0.36 ± 0.02 Hz). *Different when compared to the control microinjection of L-glutamate (P < 0.05).
compared to the baseline [from 0.36 ± 0.02 (dashed line) to 0.65 ± 0.02 Hz]. At 30 and 45 min after KYN, the pattern of responses elicited by L-glutamate was back to the control. Regarding the HR (panel B), the bradycardia produced by the microinjection of the L-glutamate into the comNTS was significantly reduced at 2 (from −83 ± 11 to −23 ± 8 bpm) and 10 min (from −83 ± 11 to −26 ± 5 bpm) after the antagonism of the ionotropic glutamate receptors with KYN microinjected into the same site in the NTS. The control increase in the tSNA (+7.5 ± 0.6%, panel C) produced by the microinjection of L-glutamate into the comNTS was almost abolished at 2 and 10 min after the microinjection of KYN into the same
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site in the NTS. The bradycardic response and the increase in tSNA in response to microinjection of Lglutamate into the comNTS were back to control levels 30 min after the microinjection of KYN. The unilateral microinjection of KYN produced no changes on the baseline of the HR, PND and tSNA. The changes in the frequency of the PND in response to L-glutamate microinjected into the NTS before and after KYN on time domain are shown in Fig. 3B.
2.3. Concentration–response curves to microinjection of trans-ACPD into the comNTS Fig. 4A shows the typical tracings of one WHBP in which microinjection of trans-ACPD (2.5 mM) was performed into the comNTS and the changes in HR, in the frequency of the PND and in the tSNA (integrated and raw) were evaluated. Panels B– D show that increasing concentrations of trans-ACPD (0.5, 1.0, 2.5, 5.0 and 10 mM) produced bradycardia (−4 ± 2, −35 ± 4, −46 ± 6, −63 ± 10 and −102 ± 9 bpm) and increase in the frequency of the PND (0.44 ± 0.02, 0.53 ± 0.04, 0.60 ± 0.02, 0.67 ± 0.03 and 0.74 ± 0.03 Hz) in a concentration-dependent manner and no major changes in the tSNA (0.22 ± 0.84%, 0.66 ± 0.32%, −1.30 ± 0.40%, 0.70 ± 0.34% and −0.62 ± 1.76%). Microinjection of the vehicle (saline) at the same level of the NTS produced negligible changes on the HR (−4 ± 2 bpm), frequency of the PND (0.37 ± 0.02 Hz) and tSNA (−1.1 ± 0.6%). The changes in the frequency of the PND in response to increasing concentrations of trans-ACPD microinjected into the NTS on time domain are shown in Fig. 6A.
2.4. Changes in the HR and frequency of the PND in response to microinjection of trans-ACPD before and after the antagonism of metabotropic glutamate receptors (MCPG, 100 mM) into the comNTS Fig. 5 summarizes the changes in the frequency of the PND (panel A) and HR (panel B) in response to microinjection of
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trans-ACPD (2.5 mM) before and at 2, 10, 30 and 45 min after the antagonism of metabotropic glutamate receptors with MCPG (100 mM) into the comNTS in the WHBP (n = 5). Microinjection of trans-ACPD (2.5 mM) produced an increase in the frequency of the PND [from 0.37 ± 0.03 (dashed line) to 0.68 ± 0.02 Hz]. The increase in the frequency of the PND produced by trans-ACPD microinjected into the comNTS was abolished at 2 [from 0.37 ± 0.02 (dashed line) to 0.36 ± 0.02 Hz] and 10 min [from 0.37 ± 0.02 (dashed line) to 0.34 ± 0.02 Hz] after antagonism of metabotropic glutamate receptors with MCPG at the same site in the NTS. At 45 min, the increase in the frequency of the PND was back to the control values. Regarding the bradycardic response produced by microinjection of trans-ACPD into the comNTS, it was significantly reduced at 2 (from −55 ± 2 to −7 ± 1 bpm), 10 (from −55 ± 2 to −5 ± 1 bpm) and 30 min (from −55 ± 2 to −25 ± 4 bpm) after the blockade of metabotropic glutamate receptors with MCPG, and it was back to the control values 45 min after the antagonism. The unilateral microinjection of MCPG produced no changes on the baseline of the HR, PND and tSNA. The changes in the frequency of the PND in response to trans-ACPD microinjected into the comNTS before and after MCPG on time domain are shown in Fig. 6B.
2.5. Changes in the HR and frequency of the PND in response to microinjection of L-glutamate before and after the simultaneous blockade of ionotropic (KYN, 250 mM) and metabotropic (MCPG, 100 mM) glutamate receptors into the comNTS Fig. 7 shows the changes in the frequency of the PND (panel A) and HR (panel B) in response to microinjection of L-glutamate (50 mM) before (control) and at 2, 10, 30 and 45 min after microinjection of both ionotropic (KYN, 250 mM) and metabotropic (MCPG, 100 mM) glutamate receptors into the commissural subnucleus of the NTS in the WHBP. Microinjection of L-glutamate produced a reduction in the frequency
Fig. 3 – (A) Changes in the frequency of the PND in the 50 s subsequent to microinjections of increasing concentrations of L-glutamate into the comNTS. (B) Changes in the frequency of the PND in the 50 s subsequent to microinjection of L-glutamate (50 mM) into the comNTS before and after KYN (250 mM). *Different when compared to saline (A) or control microinjection of L-glutamate (B) (P < 0.05).
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Fig. 4 – Effects of microinjection of trans-ACPD microinjected into the comNTS on heart rate (HR), frequency of phrenic nerve discharge (PND) and thoracic sympathetic nerve activity (tSNA). Panel A illustrates a group of tracings from a representative WHBP showing the raw tracings of ECG and PND, the changes in HR, ∫ PND, ∫ tSNA and the raw tSNA in response to microinjection of trans-ACPD (2.5 mM) into the comNTS. Panels B–D represent the changes in the heart rate ( ΔHR, bpm), in the frequency of the PND (Hz) and in the tSNA ( Δ%), respectively, in response to microinjection of increasing concentrations of trans-ACPD into the comNTS (0.5, 1, 2.5, 5 and 10 mM in 20 nl; n = 7).
of the PND (from 0.39 ± 0.03 to 0.07 ± 0.03 Hz), and at 2 and 10 min after the double blockade, the changes in the frequency of PND were almost abolished (from 0.47 ± 0.03 to 0.58 ± 0.04 Hz and from 0.49 ± 0.03 to 0.50 ± 0.06 Hz, respectively). At 30 and 45 min after the double blockade (KYN + MCPG), the reduction in the frequency of the PND in response to microinjection of L-glutamate into the comNTS was back to the control. With respect to the bradycardic response produced by microinjection of L-glutamate into the comNTS, it was also significantly reduced at 2 (from −76 ± 7 to −19 ± 5 bpm) and 10 min (from −76 ± 7 to −18 ± 3 bpm) after the double blockade. This pattern of attenuation of the magnitude of the bradycardic response was similar to the attenuation produced by the blockade of ionotropic receptors with KYN (Fig. 2B). The unilateral microinjection of KYN + MCPG produced no changes on the baseline of the HR, PND and tSNA. The changes in frequency of PND in response to L-glutamate microinjected into the NTS before
and after the double blockade (KYN + MCPG) on time domain are shown in Fig. 8.
2.6.
Histology
Fig. 9A presents a photomicrography of a coronal section of the brainstem of one WHBP representative of the groups showing the track of the micropipette in the direction of the comNTS and also the center of the microinjection. Fig. 9B is a diagram of a coronal section of the medulla showing the sites of microinjections of L-glutamate into the comNTS of a group of 9 WHBPs.
3.
Discussion
The experimental approach of microinjection into the NTS of awake rats has the advantage of being performed in an
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Fig. 5 – (A) Changes in the frequency of PND (Hz) in response to microinjection of trans-ACPD (2.5 mM) before and at 2, 10, 30 and 45 min after the blockade of metabotropic glutamate receptors with MCPG (100 mM) into the comNTS (n = 5). (B) Changes in the heart rate ( ΔHR) in response to the same microinjection. Dashed line indicates the baseline of the frequency of PND (0.37 ± 0.03 Hz). *Different when compared to the control microinjection of trans-ACPD (P < 0.05).
anesthetic-free neural environment, whereas the experiments performed in anesthetized rats have the advantage of the use of glass micropipettes, which allows microinjections of volumes smaller than those obtained with the needles used in awake rats. It is important to consider that under anesthesia, the magnitude of the cardiovascular and respiratory responses to microinjections into the NTS or to activation of different reflexes may vary (Machado and Bonagamba, 1992). In the present study, we used a decerebrated artificially perfused preparation (Paton, 1996), which presents the following advantages: (a) cardiovascular reflexes and eupneic respiratory pattern are preserved; (b) activities from the sympathetic and phrenic nerves can be recorded simultaneously; (c) small volumes of microinjections can be performed into the NTS under direct microscopic observation scope; and (d) the effects of anesthetics are prevented. On the other hand, this preparation also presents some disadvantage, such as the loss of descending influences from the midbrain and higher centers due to the decerebration.
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In the present study, performed in an in situ unanesthetized preparation, we observed several important findings with respect to the autonomic and respiratory responses related to activation of ionotropic and metabotropic glutamate receptors in the comNTS caudal to the CS. Among these findings, we highlight the following: (a) in the WHBP, the microinjection of L-glutamate (50 mM, 20 nl) into the comNTS produced bradycardia, increase in thoracic sympathetic nerve activity and reduction in the frequency of the PND; (b) both bradycardia and increase in tSNA in response to L-glutamate were attenuated by previous microinjection of kynurenic acid (250 mM, 20 nl) into the same site in the NTS; (c) the reduction in the frequency of the PND produced by microinjection of L-glutamate into the comNTS was not only blocked by kynurenic acid but also reversed to an increase in the frequency of the PND; (d) the increase in the frequency of the PND produced by L-glutamate microinjected into the comNTS after blockade of the ionotropic glutamate receptors was abolished after microinjection of MCPG (100 mM, 20 nl) into the same site in the NTS, indicating that metabotropic receptors are involved in the observed increase in the frequency of the PND; and (e) the microinjection of increasing doses of a metabotropic glutamate receptor agonist (trans-ACPD) produced increase in the frequency of the PND in a concentration-dependent manner, supporting the concept that metabotropic glutamate receptors are involved in the in the increase in the frequency of the PND. Our data support the conclusion that microinjection of Lglutamate into the comNTS activates mainly ionotropic glutamate receptors. First, the response of L-glutamate was significantly attenuated by previous NTS administration of kynurenic acid, a selective ionotropic glutamate receptor antagonist, a pattern of response similar to those observed in previous studies (Bertolino et al., 1989; Leone and Gordon, 1989; Andresen and Kunze, 1994). Second, several studies demonstrated that microinjection of selective ionotropic glutamate receptor agonists into the NTS produce responses similar to those produced by L-glutamate, and also that these responses were inhibited by kynurenic acid (Kubo and Kihara, 1991; Leone and Gordon, 1989; Le Galloudec et al., 1989; Talman, 1989; Almado and Machado, 2005). Our data show that L-glutamate acting on ionotropic glutamate receptors in the comNTS caudal to CS produces bradycardia, increase in tSNA and reduction in the frequency of the PND. The autonomic responses elicited by microinjection of Lglutamate into the comNTS in the present study (sympathoexcitation and bradycardia) were similar to the pattern of responses elicited by chemoreflex activation in the WHBP (Antunes et al., 2005), nevertheless, with less intensity. In addition, these autonomic responses were attenuated by KYN microinjected into the same site in the NTS indicating a role for the ionotropic glutamate receptors. Although these responses are chemoreflex-like, these data do not allow us to suggest that L-glutamate is exclusively acting on neurons involved in the processing of the chemoreflex pathways at the comNTS level. In fact, the sites shown in Fig. 9 suggest that medial portions of the regions from
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Fig. 6 – (A) Changes in the frequency of the PND in the 50 s subsequent to microinjections of increasing concentrations of trans-ACPD into the comNTS. (B) Changes in the frequency of the PND in the 50 s subsequent to microinjection of trans-ACPD (2.5 mM) into the comNTS before and after MCPG (100 mM). *Different when compared to saline (A) or control microinjection of trans-ACPD (B) (P < 0.05).
which baroreceptor-like responses are elicited by L-glutamate may have also contributed to the observed responses in this study. For this purpose, further studies using ionotropic glutamate receptors antagonists against the chemoreflex activation itself are required to evaluate this interesting possibility. The reduction in the frequency of the PND produced by microinjection of L-glutamate into the comNTS may be related to the activation of neurons involved in the Hering– Breuer reflex pathways identified as “pump cells” (Bonham and McCrimmon, 1990; Bonham et al., 1993; Berger et al., 1995). The activation of these neurons produces a reduction in the frequency of the PND that seems to be mediated by ionotropic glutamate receptors (Bonham et al., 1993; Rybak et al., 2004). The involvement of ionotropic glutamate receptors in the reduction in the frequency of the PND produced by microinjection of L-glutamate into the comNTS is strongly supported by the fact that the microinjection of kynurenic acid into the same site in the NTS blocked this response. In addition to the activation of ionotropic receptors, L-glutamate also activates metabotropic receptors in the comNTS, producing an increase in the frequency of the PND as observed when L-glutamate was microinjected after blockade of ionotropic glutamate receptors with KYN. This increase in the frequency of the PND was blocked when metabotropic glutamate receptor antagonist (MCPG) was combined with KYN. The involvement of metabotropic receptors in the increase in the frequency of the PND to microinjection of L-glutamate after the blockade of ionotropic receptors with kynurenic acid is also supported by the data from the experimental protocol using increasing doses of trans-ACPD, which clearly demonstrated that the activation of metabotropic receptors produced a dose-dependent increase in the frequency of the PND.
In conclusion, microinjection of L-glutamate into the comNTS caudal to CS activates ionotropic receptors producing sympathoexcitation, whereas the bradycardic response to L-glutamate seems to be mediated by both ionotropic and metabotropic receptors activation. The most exciting finding of the present study is related to the changes in the frequency of the PND because L-glutamate activating ionotropic receptors produces reduction in the frequency of the PND whereas the activation of metabotropic receptors produces increase in the frequency of the PND. Considering that our goal was to characterize the autonomic and respiratory response to microinjection of L-glutamate into the comNTS caudal to CS, the data obtained in the present study open an interesting field to explore the involvement of L-glutamate and its ionotropic and metabotropic receptors in the neurotransmission and neuromodulation of the autonomic and respiratory components of the cardiovascular reflexes in the commissural subnucleus of the NTS.
4.
Experimental procedure
4.1.
General surgical procedures
The experiments were performed in an in situ unanesthetized decerebrated WHBP as described previously by Paton (1996). Male Wistar rats (70–90 g) were anesthetized deeply with halothane and the level of anesthesia was assessed by absence of response to a noxious pinch of either the paw or the tail. Following subdiaphragmatic transection, the rostral half of the animal was submerged in cooled artificial cerebrospinal fluid (ACSF) carbogen gassed (95% O2 and 5% CO2), decerebrated at the precollicular level and skinned, the
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Medicine approved December Brazilian (COBEA).
4.2.
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of Ribeirão Preto, University of São Paulo, all procedures during the ordinary meeting on 1, 2003, according to recommendations of the Association for Laboratory Animal Science
Recordings of electrocardiogram and nerve activities
Left phrenic nerve activity was recorded from its central end using a glass suction electrode held in a micromanipulator. Rhythmic ramping phrenic nerve discharge (PND) gave a continuous physiological index of the preparation viability. The electrocardiogram (ECG) was visible on the phrenic nerve recording, which allowed us evaluate the heart rate (HR) by using a low-pass filter (Figs. 1A and 4A). Sympathetic nerve activity was recorded from the thoracic sympathetic chain (tSNA) at the level of T5–T10 using a second glass suction bipolar electrode. Signals were AC amplified, band-pass filtered (8 Hz to 3 kHz) and displayed on a computer using the software Spike 2 (Cambridge Electronic Design, Cambridge, England).
4.3.
Fig. 7 – (A) Changes in the frequency of PND (Hz) in response to microinjection of L-glutamate (50 mM) before and at 2, 10, 30 and 45 min after the double blockade of ionotropic and metabotropic glutamate receptors with kynurenic acid (KYN, 250 mM) combined with MCPG (100 mM) into the comNTS (n = 6). (B) Changes in the heart rate ( ΔHR) in response to the same microinjection. Dashed line indicates the baseline of the frequency of PND (0.47 ± 0.03 Hz at 2 min after the blockade). *Different when compared to the control microinjection of L-glutamate (P < 0.05).
Microinjections into the NTS
The calamus scriptorius (CS) was used as a landmark for microinjections into the comNTS. Drugs were applied unilaterally via a three-barreled micropipette (tip diameter 20–30 μm). The tip of the micropipette was driven into the medulla to a depth of 0.3–0.4 mm ventral to the dorsal surface, 0.2–0.4 mm caudal relative to CS and between 0.25 and 0.35 mm from midline. The injected volume for all drugs (20 nl) was determined by meniscus calibration in the eyepiece of the microscopy. At the end of the experiments, the brain was removed, fixed and histological procedures were performed in order to verify the micropipette track and the center of microinjections in the comNTS.
4.4.
Agonists and antagonists microinjected into the NTS
L-Glutamate,
descending aorta was then isolated, the heart exposed by removal of the left ribs and the lungs removed. The dorsal surface of the brainstem was exposed by removal of the occipital bone and cerebellum. The WHBP was moved to a recording chamber, then the descending aorta was cannulated and perfused retrogradely with ACSF (in mM): NaCl, 125; NaHCO3, 24; KCl, 5; CaCl2, 2.5; MgSO4, 1.25; KH2PO4, 1.25; dextrose, 10; oncotic agent (Ficoll® 70, 1.25%; Sigma, St. Louis, MO, USA) using a roller pump (Watson-Marlow 502s, Falmouth, Cornwall, UK) via double lumen cannula. A neuromuscular blocker (vecuronium bromide, 0.04 mg/ ml; Norcuron Organon Teknika, Brazil) was used for stopping the spontaneous chest wall respiratory movements. Perfusion pressure was maintained in a narrow range (from 50 to 70 mm Hg) by adjusting flow rate of the perfusion pump. The perfusate was gassed with carbogen continuously, warmed to 32 °C and filtered using a nylon mesh (pore size: 25 μm; Millipore, Ireland). The Ethical Committee for Animal Experimentation of the School of
kynurenic acid (KYN), trans-1-amino-1,3-cyclopentanediocarboxylic acid (trans-ACPD) and (±)-α-methyl-4carboxyphenylglycine (MCPG) were obtained from Sigma (St. Louis, MO, USA). Drugs were dissolved in saline (NaCl 0.9%) and adjusted to pH 7.4 by adding NaHCO3. Concentrations are expressed as the free base of each drug. The concentration of 250 mM of KYN was the concentration that attenuated all the responses to L-glutamate microinjection into the NTS (sympathoexcitation, bradycardia and reduction in the frequency of PND) without affecting the baseline of the parameters evaluated. In a preliminary study, we tested two other concentrations of KYN (50 and 100 mM). These concentrations produced no changes in the sympathoexcitation and in the reduction in the phrenic nerve discharge elicited by microinjection of Lglutamate into the NTS [50 mM: tSNA (+13 ± 3 vs. +11 ± 4%) and PND (from 0.36 ± 0.02 to 0.11 ± 0.02 Hz vs. 0.35 ± 0.03 to 0.33 ± 0.02 Hz); and 100 mM: tSNA (+12 ± 2 vs. +13 ± 3%) and PND (from 0.35 ± 0.03 to 0.09 ± 0.04 Hz vs. 0.36 ± 0.04 to 0.35 ± 0.03 Hz)]. The
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n = 4). The concentration of MCPG (100 mM) was used in accordance with previous studies from our laboratory in which this concentration was effective in blocking the responses elicited by microinjection of trans-ACPD in the NTS (Antunes and Machado, 2003).
4.5.
Fig. 8 – Changes in the frequency of the PND in the 50 s subsequent to microinjection of L-glutamate (50 mM) into the comNTS before and after KYN (250 mM) combined with MCPG (100 mM). *Different when compared to the control microinjection of L-glutamate (P < 0.05). concentration of 100 mM of KYN produced only a significant reduction in the bradycardia produced by the microinjection of L-glutamate (−72 ± 6 vs. −42 ± 7 Δbpm,
Data analysis
All data were analyzed off-line using Spike2 software with custom-written scripts. Baseline and peak responses in HR and tSNA were measured. Frequency of bursts of the PND was measured in a time-dependent way (counted each 10 s starting 40 s before the microinjection and ending 50 s after microinjection). Besides, we evaluated the peak of the response (frequency of PND) after each microinjection. The rectified and integrated signal of the tSNA (100 ms time constant) was measured for a period covering 10 s before and 10s after each microinjection as previously described (Braga and Machado, 2006). The data from tSNA were expressed as percentage of the control. The significance of effects was assessed by twoway ANOVA followed by Tukey's post hoc test (P < 0.05) to evaluate the time course responses in the frequency of PND and one-way ANOVA followed by Tukey's post hoc test (Pstandard error (SE) and n is the number of preparations.
Fig. 9 – (A) Photomicrography of a transverse section of the brainstem of one rat, representative of the group, showing the unilateral microinjection site in the commissural subnucleus of the NTS. The arrow indicates the center of the microinjection in the NTS. Scale bar corresponds to 500 μm. (B) Diagram of a coronal section of the medulla showing the site of microinjections of L-glutamate into the comNTS caudal to CS (n = 9). CC, central canal; 10 dorsal motor nucleus, 12 hypoglossal nucleus.
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Acknowledgments The authors thank Prof. Julian F.R. Paton, University of Bristol, UK, and Prof. Jeffrey T. Potts, University of Missouri, EUA, for their intellectual and material support to the development of the WHBP in our laboratory. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2001/11190-8 and 2004/03285-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ, 472704/ 2004-4).
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